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1.
Nat Commun ; 11(1): 4289, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855397

RESUMO

Older organs represent an untapped potential to close the gap between demand and supply in organ transplantation but are associated with age-specific responses to injury and increased immunogenicity, thereby aggravating transplant outcomes. Here we show that cell-free mitochondrial DNA (cf-mt-DNA) released by senescent cells accumulates with aging and augments immunogenicity. Ischemia reperfusion injury induces a systemic increase of cf-mt-DNA that promotes dendritic cell-mediated, age-specific inflammatory responses. Comparable events are observed clinically, with the levels of cf-mt-DNA elevated in older deceased organ donors, and with the isolated cf-mt-DNA capable of activating human dendritic cells. In experimental models, treatment of old donor animals with senolytics clear senescent cells and diminish cf-mt-DNA release, thereby dampening age-specific immune responses and prolonging the survival of old cardiac allografts comparable to young donor organs. Collectively, we identify accumulating cf-mt-DNA as a key factor in inflamm-aging and present senolytics as a potential approach to improve transplant outcomes and availability.


Assuntos
DNA Mitocondrial/efeitos adversos , Dasatinibe/farmacologia , Inflamação/prevenção & controle , Transplante de Órgãos/métodos , Quercetina/farmacologia , Adulto , Envelhecimento/fisiologia , Animais , Diferenciação Celular , Ácidos Nucleicos Livres , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Citocinas/metabolismo , DNA Mitocondrial/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Transplante de Coração/efeitos adversos , Transplante de Coração/métodos , Humanos , Inflamação/etiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Transplante de Órgãos/efeitos adversos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Doadores de Tecidos
2.
J Pharmacol Sci ; 144(2): 69-75, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32713799

RESUMO

The mechanism of the papaverine (PV) for the treatment of cerebral ischemia remains unclear. A total of 42 mice induced with focal cerebral ischemia were randomly divided into three groups: PV,baicalin (BA)and vehicle group. Both PV and BA could significantly reduce the ischemic infarct volume (P < 0.05). Pathway enrichment analysis was performed on MetaCore™ to search the molecular pathways associated with the gene expression profile of PV, compared with vehicle and BA. Compared with vehicle, we found that 60% of the top 10 pathways in PV group were related to immune response. The top 10 biological processes of the targeted pathways were mainly related to the multiple immunomodulatory process of neuro-vascular inflammation, including immune_Th17-deried cytokins, regulation of angiogenesis, cell adhesion_Leucocyte chemotaxis, antigen presentation, cell adhesion_synaptic contact, and inflammation related to Amphoterin signaling. Moreover, compared with BA, 17 pathways of PV were identified, and 58.82% (10/17) were also related to immune response, especially, half of the top 10 pathways with the lower p-value. In these top 10 pathways, 4 were the cytokine-mediated signaling pathways, which play key role in inflammation, like IL-17 signaling pathways with the activation of G-CSF,IL-23,RANKL, p38MAPK and NF-κB.These findings indicate that PV may be an efficacious pluripotent anti-inflammatory agent against cerebral ischemic-reperfusion injury by targeting on multiple immunomodulatory process of neuro-vascular inflammation.


Assuntos
Anti-Inflamatórios , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Fatores Imunológicos , Papaverina/farmacologia , Papaverina/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/genética , Animais , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Inflamação , Interleucina-17/genética , Interleucina-17/metabolismo , Masculino , Camundongos Endogâmicos , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 205-211, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389167

RESUMO

Objective To investigate the effects of phosphate and tension homology deleted on chromsome ten (PTEN) knockout on rat heart function and pyroptosis of cardiomyocytes mediated by NLR family pyrin domain containing 3 (NLRP3). Methods Rat models of myocardial ischemia/reperfusion (I/R) injury were established. The rats were divided into sham operation group (wild-type healthy rats), wild-type I/R group (wild-type healthy rats treated with myocardial I/R), and I/R group (PTEN KO rats treated with myocardial I/R). PTEN mRNA level was detected by reverse transcription PCR, and myocardial pathological damage was observed by HE staining. Heart rate (HR) and left ventricular wall thickness (LVWT) were measured by echocardiography, and left ventricular systolic blood pressure (LVSP), left ventricular ejection fraction (LVEF), and fraction shortening (FS) were recorded by BL-420F bioassay system. Serum creatine kinase isoenzyme (CK-MB), myoglobin (Mb) and cardiac troponin I (cTnI) were detected by ELISA. Western blot analysis was used to detect the protein expression of NLRP3, embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1), caspase-1 (caspase-1), and IL-1ß in heart tissues. Immunohistochemical staining was performed to detect the content of caspase-1 in cardiac tissues. Apoptosis of myocardial tissue was observed with TUNEL staining. Results Compared with the sham operation group, PTEN mRNA and protein levels in the wild-type I/R group significantly increased, HR, LVSP, LVEF, FS, and LVWT went down significantly, and serum CK-MB, Mb, and cTnI levels significantly increased, and NLRP3, ELAVL1, caspase-1, and IL-1ß protein expression levels went up significantly. After PTEN was knocked out, PTEN mRNA and protein levels were significantly reduced, the pathological damage of cardiomyocytes was alleviated, and HR, LVSP, LVEF, FS, and LVWT were significantly elevated, and serum CK-MB, Mb, and cTnI levels were significantly inhibited. NLRP3, ELAVL1, caspase-1, and IL-1ß protein levels and the number of apoptotic cardiomyocytes were significantly reduced after PTEN knockout. Conclusion Knockout of PTEN can alleviate the pathological damage of myocardium and inhibit nlrp3-mediated apoptosis of cardiomyocytes, indicating that knockout of PTEN can alleviate myocardial I/R damage.


Assuntos
Isquemia Miocárdica/genética , Miócitos Cardíacos/citologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , PTEN Fosfo-Hidrolase/genética , Piroptose , Traumatismo por Reperfusão/genética , Animais , Técnicas de Inativação de Genes , Ratos , Volume Sistólico , Função Ventricular Esquerda
4.
Am J Physiol Renal Physiol ; 318(6): F1531-F1538, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390514

RESUMO

Renal ischemia-reperfusion injury (I/R) usually occurs in renal transplantation and partial nephrectomy, which could lead to acute kidney injury. However, the effective treatment for renal I/R still remains limited. In the present study, we investigated whether inhibition of chromobox 7 (CBX7) could attenuate renal I/R injury in vivo and in vitro as well as the potential mechanisms. Adult male mice were subjected to right renal ischemia and reperfusion for different periods, both with and without the CBX7 inhibitor UNC3866. In addition, human kidney cells (HK-2) were subjected to a hypoxia/reoxygenation (H/R) process for different periods, both with or without the CBX7 inhibitor or siRNA for CBX7. The results showed that expression of CBX7, glucose regulator protein-78 (GRP78), phosphorylated eukaryotic translation initiation factor-2α (p-eIF2α), and C/EBP homologous protein (CHOP) were increased after extension of I/R and H/R periods. Moreover, overexpression of CBX7 could elevate the expression of CBX7, GRP78, p-eIF2α, and CHOP. However, CBX7 inhibition with either UNC3866 or genetic knockdown led to reduced expression of GRP78, p-eIF2α, and CHOP through nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 activation in I/R and H/R injury. Furthermore, ML385, the Nrf2 inhibitor, could elevate endoplasmic reticulum stress levels, abrogating the protective effects of UNC3866 against renal I/R injury. In conclusion, our results demonstrated that CBX7 inhibition alleviated acute kidney injury by preventing endoplasmic reticulum stress via the Nrf2/HO-1 pathway, indicating that CBX7 inhibitor could be a potential therapeutic target for renal I/R injury.


Assuntos
Lesão Renal Aguda/prevenção & controle , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Rim/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oligopeptídeos/farmacologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Lesão Renal Aguda/enzimologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Hipóxia Celular , Linhagem Celular , Heme Oxigenase-1/genética , Humanos , Rim/enzimologia , Rim/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Fator 2 Relacionado a NF-E2/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
5.
Biol Res ; 53(1): 17, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312329

RESUMO

BACKGROUND: Inflammation and apoptosis are considered to be two main factors affecting ischemic brain injury and the subsequent reperfusion damage. MiR-19a-3p has been reported to be a possible novel biomarker in ischemic stroke. However, the function and molecular mechanisms of miR-19a-3p remain unclear in cerebral ischemia/reperfusion (I/R) injury. METHODS: The I/R injury model was established in vivo by middle cerebral artery occlusion/reperfusion (MCAO/R) in rats and in vitro by oxygen-glucose deprivation and reperfusion (OGD/R) induced SH-SY5Y cells. The expression of miR-19a-3p was determined by reverse transcription quantitative PCR. The infarction volumes, Neurological deficit scores, apoptosis, cell viability, pro-inflammatory cytokines and apoptosis were evaluated using Longa score, Bederson score, TTC, TUNEL staining, CCK-8, ELISA, flow cytometry assays. Luciferase reporter assay was utilized to validate the target gene of miR-19a-3p. RESULTS: We first found miR-19a-3p was significantly up-regulated in rat I/R brain tissues and OGD/R induced SH-SY5Y cells. Using the in vivo and in vitro I/R injury model, we further demonstrated that miR-19a-3p inhibitor exerted protective role against injury to cerebral I/R, which was reflected by reduced infarct volume, improved neurological outcomes, increased cell viability, inhibited inflammation and apoptosis. Mechanistically, miR-19a-3p binds to 3'UTR region of IGFBP3 mRNA. Inhibition of miR-19a-3p caused the increased expression of IGFBP3 in OGD/R induced SH-SY5Y cells. Furthermore, we showed that IGFBP3 overexpression imitated, while knockdown reversed the protective effects of miR-19a-3p inhibitor against OGD/R-induced injury. CONCLUSIONS: In summary, our findings showed miR-19a-3p regulated I/R-induced inflammation and apoptosis through targeting IGFBP3, which might provide a potential therapeutic target for cerebral I/R injury.


Assuntos
Isquemia Encefálica/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , MicroRNAs/genética , Oxigênio/metabolismo , Traumatismo por Reperfusão/genética , Animais , Apoptose , Biomarcadores/metabolismo , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Glucose/deficiência , Masculino , Neurônios/metabolismo , Neuroproteção , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral , Regulação para Cima
6.
J Stroke Cerebrovasc Dis ; 29(6): 104801, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32249206

RESUMO

BACKGROUND: Ischemic stroke is the leading cause of disability and death globally. Micro-RNAs (miRNAs) have been reported to play important roles in the development and pathogenesis of the nervous system. However, the exact function and mechanism of miRNAs have not been fully elucidated about brain damage caused by cerebral ischemia/reperfusion (I/R). METHODS: In this study, we explored the neuroprotective effects of miR-219a-5p on brain using an in vitro ischemia model (mouse neuroblastoma N2a cells treated with oxyglucose deprivation and reperfusion), and in vivo cerebral I/R model in mice. Western blot assay and Reverse Transcription-Polymerase Chain Reaction were used to check the expression of molecules involved. Flow cytometry and cholecystokinin were used to examine cell apoptosis, respectively. RESULTS: Our research shows that miR-219a-5p gradually decreases in cerebral I/R models in vivo and in vitro. In vitro I/R, we find that miR-219a-5p mimics provided evidently protection for cerebral I/R damage, as shown by increased cell viability and decreased the release of LDH and cell apoptosis. Mechanically, our findings indicate that miR-219a-5p binds to cAMP specific 3', 5'-cyclic phosphodiesterase 4D (PDE4D) mRNA in the 3'-UTR region, which subsequently leads to a decrease in Pde4d expression in I/R N2a cells. CONCLUSIONS: Our results provide new ideas for the study of the mechanism of cerebral ischemia/reperfusion injury, and lay the foundation for further research on the treatment of brain I/R injury. Upregulation of miR-219a-5p decreases cerebral ischemia/reperfusion injury by targeting Pde4d in vitro.


Assuntos
Apoptose , Encéfalo/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Infarto da Artéria Cerebral Média/enzimologia , MicroRNAs/metabolismo , Neurônios/enzimologia , Traumatismo por Reperfusão/enzimologia , Regiões 3' não Traduzidas , Animais , Sítios de Ligação , Encéfalo/patologia , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurônios/patologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais
7.
Life Sci ; 249: 117517, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32147431

RESUMO

AIM: To explore the role and mechanism of Hydrogen peroxide-inducible clone-5 (Hic-5) in hepatic ischemia reperfusion injury. METHODS: Hic-5 KO and WT mice were used to establish the liver ischemia reperfusion model (HI/R). Primary hepatocytes were isolated to establish hypoxic reoxygenation model (H/R). AST and ALT were measured by automatic biochemical analyzer. Liver tissue sections were stained with HE and Tunnel. RNA and proteins were extracted from liver tissues, and expressions of Il-6, Il-10, CCL-2, CXCL-10, P65, Caspase-3, TLR4 and FADD were detected at gene and protein levels. Liver cell apoptosis was detected by flow cytometry and immunofluorescence. Primary hepatocytes were stimulated by LPS to establish a model of hepatocyte apoptosis, and cell inflammation and apoptosis-related proteins were detected. RESULTS: After HI/R, ALT and AST in serum were up-regulated, some hepatocyte apoptosis were observed in pathological sections. Hic-5 expression was increased in WT mice after HI/R, and liver damage were severer than KO mice. The expression of IL-6, CCL-2 and CXCL-10 in the liver of KO mice was low, and the expression of IL-10 was high. Further studies showed that KO mice showed lower expression of P65, Caspase3 and TLR4. In H/R model, hepatocytes also showed the same trend. Finally, after LPS stimulation, the results showed that the inflammation and apoptosis induced by LPS were significantly reduced in Hic-5 knocked hepatocytes. CONCLUSION: Hic-5 was found to promote inflammation through NF-kb signaling pathway and apoptosis through TLR4-FADD signaling pathway in mice with HI/R, thus aggravating liver injury in mice.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Proteínas com Domínio LIM/genética , Fígado/irrigação sanguínea , NF-kappa B/metabolismo , Traumatismo por Reperfusão/genética , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
8.
Gene ; 741: 144562, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32169629

RESUMO

Renal Ischemia/Reperfusion (rI/R)-induced acute lung injury (ALI) is a major problem in rI/R. The objective of the current study was to explore the defensive roles of propofol (Pro), an intravenous anesthetic, on rI/R-induced ALI through mitogen-activated protein kinase (MAPK) signaling. Rats were divided into Sham, Pro (10 mg/kg), rI/R, rI/R + Pro (5 mg/kg), and rI/R + Pro (10 mg/kg) groups. Rats were treated with Pro at 1 h after rI/R treatment. Serum and lung tissues at 24 h after rI/R were collected to evaluate morphological changes and the expression of myeloperoxidase (MPO), inflammatory cytokines, and crucial proteins in the MAPK pathway. Pro attenuated the production of mediators, resulting in reduced levels of autophagy and apoptosis by restricting the MAPK pathway in rI/R-induced ALI model. Pro represses rI/R-induced pulmonary autophagy and apoptosis by decreasing the production of inflammatory molecules, and the effects of Pro are involved in the inhibition of the MAPK pathway.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/tratamento farmacológico , Propofol/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Lesão Renal Aguda/complicações , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/genética , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ratos , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
9.
J Biomed Sci ; 27(1): 40, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32138732

RESUMO

BACKGROUND: The present study aimed to verify whether long noncoding RNA (lncRNA) MALAT1 is involved in brain tissue damage induced by ischemia-reperfusion injury, and to explore the mechanism by which MALAT1 regulates aquaporin 4 (AQP4). METHODS: In this study, we established glucose deprivation (OGD)/reoxygenation (RX) astrocyte cell model and middle cerebral artery occlusion (MCAO)/reperfusion mouse model in vitro and in vivo. Then cell counting kit-8 assay, flow cytometry analysis, Triphenyltetrazolium chloride (TTC) staining, and western blotting were used to determine cell viability, cell apoptosis, cerebral infarction volume, and the abundance of AQP4, respectively. RESULTS: We found that the level of MALAT1 was significantly upregulated in both the MCAO/reperfusion model and OGD/RX model. Knockdown of MALAT1 increased cell viability and reduced cell apoptosis in MA-C cells, while an AQP4 siRNA combined with a siRNA targeting MALAT1 could not enhance this effect. Further experiments showed that MALAT1 positively regulated AQP4 expression via miR-145. The MALAT1 siRNA did not alleviate the exacerbation of damage after miR-145 inhibitor action. However, an miR-145 inhibitor reversed the protection effects of MALAT1, indicating that MALAT1 silencing protects against cerebral ischemia-reperfusion injury through miR-145. TTC staining showed that the infracted area of whole brain was significantly attenuated in treated with sh-MALAT1 group in vivo. CONCLUSION: Taken together, our study confirmed that MALAT1 promotes cerebral ischemia-reperfusion injury by affecting AQP4 expression through competitively binding miR-145, indicating that MALAT1 might be a new therapeutic target for treatment cerebral ischemic stroke.


Assuntos
Aquaporina 4/genética , Regulação da Expressão Gênica , Inativação Gênica , MicroRNAs/genética , RNA Longo não Codificante/genética , Traumatismo por Reperfusão/genética , Animais , Aquaporina 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Regulação para Cima
10.
Am J Physiol Renal Physiol ; 318(5): F1100-F1112, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32116018

RESUMO

In the early proximal tubule, Na+-glucose cotransporter 2 (SGLT2) mediates the bulk of renal glucose reabsorption. Gene deletion in mice (Sglt2-/-) was used to determine the role of SGLT2 in acute kidney injury induced by bilateral ischemia-reperfusion (IR). In Sglt2-/- and littermate wild-type mice, plasma creatinine increased similarly on day 1 after IR. This was associated with an equal increase in both genotypes in the urinary kidney injury molecule-1-to-creatinine ratio, a tubular injury marker, and similarly reduced urine osmolality and increased plasma osmolality, indicating impaired urine concentration. In both IR groups, FITC-sinistrin glomerular filtration rate was equally reduced on day 14, and plasma creatinine was similarly and incompletely restored on day 23. In Sglt2-/- mice subjected to IR, fractional urinary glucose excretion was increased on day 1 but reduced and associated with normal renal Na+-glucose cotransporter 1 (Sglt1) mRNA expression on day 23, suggesting temporary SGLT1 suppression. In wild-type mice subjected to IR, renal Sglt1 mRNA was likewise normal on day 23, whereas Sglt2 mRNA was reduced by 57%. In both genotypes, IR equally reduced urine osmolality and renal mRNA expression of the Na+-K+-2Cl- cotransporter and renin on day 23, suggesting thick ascending limb dysfunction, and similarly increased renal mRNA expression of markers of injury, inflammation, oxidative stress, and fibrosis (kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, monocyte chemoattractant protein-1, transforming growth factor-ß1, NADPH oxidase-2, and collagen type 1). This was associated with equal increases in kidney histological damage scores and similar degree of capillary loss in both genotypes. The data indicate that genetic deletion of SGLT2 did not protect the kidneys in the initial injury phase or the subsequent recovery phase in a mouse model of IR-induced acute kidney injury.


Assuntos
Lesão Renal Aguda/metabolismo , Glicemia/metabolismo , Rim/metabolismo , Traumatismo por Reperfusão/metabolismo , Transportador 2 de Glucose-Sódio/deficiência , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Lesão Renal Aguda/fisiopatologia , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Taxa de Filtração Glomerular , Rim/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Eliminação Renal , Reabsorção Renal , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Transportador 2 de Glucose-Sódio/genética , Fatores de Tempo
11.
Am J Physiol Renal Physiol ; 318(5): F1147-F1159, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174142

RESUMO

Meprin metalloproteases have been implicated in the progression of kidney injury. Previous work from our group has shown that meprins proteolytically process the catalytic subunit of protein kinase A (PKA-C), resulting in decreased PKA-C kinase activity. The goal of the present study was to determine the PKA-C isoforms impacted by meprin-ß and whether meprin-ß expression affects downstream mediators of the PKA signaling pathway in ischemia-reperfusion (IR)-induced kidney injury. IR was induced in 12-wk-old male wild-type (WT) and meprin-ß knockout (ßKO) mice. Madin-Darby canine kidney cells transfected with meprin-ß cDNA were also subjected to 2 h of hypoxia. Western blot analysis was used to evaluate levels of total PKA-C, PKA-Cα, PKA-Cß, phosphorylated (p-)PKA-C, and p-ERK1/2. Meprin-ß expression enhanced kidney injury as indicated by levels of neutrophil gelatinase-associated lipocalin and cystatin C. IR-associated decreases were observed in levels of p-PKA-C in kidney tissue from WT mice but not ßKO mice, suggesting that meprin-ß expression/activity is responsible for the in vivo reduction in kinase activity. Significant increases in levels of PKA-Cß were observed in kidney lysates for WT mice but not ßKO mice at 6 h post-IR. Proximal tubule PKA-Cß increases in WT but not ßKO kidneys were demonstrated by fluorescent microscopy. Furthermore, IR-induced injury was associated with significant increases in p-ERK levels for both genotypes. The present data demonstrate that meprin-ß enhances IR-induced kidney injury in part by modulating mediators of the PKA-Cß signaling pathway.


Assuntos
Lesão Renal Aguda/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Rim/enzimologia , Metaloendopeptidases/metabolismo , Traumatismo por Reperfusão/enzimologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Hipóxia Celular , Modelos Animais de Doenças , Cães , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Rim/patologia , Células Madin Darby de Rim Canino , Masculino , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
12.
Stroke ; 51(3): 975-985, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32078472

RESUMO

Background and Purpose- Microglia/macrophages (Mi/MΦ) can profoundly influence stroke outcomes by acquiring functionally dominant phenotypes (proinflammatory or anti-inflammatory; deleterious or salutary). Identification of the molecular mechanisms that dictate the functional status of Mi/MΦ after brain ischemia/reperfusion may reveal novel therapeutic targets for stroke. We hypothesized that activation of TAK1 (transforming growth factor beta-activated kinase 1), a key MAP3K upstream of multiple inflammation-regulating pathways, drives Mi/MΦ toward a proinflammatory phenotype and potentiates ischemia/reperfusion brain injury. Methods- Young adult mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) followed by reperfusion. TAK1 was targeted by tamoxifen-induced Mi/MΦ-specific knockout or administration of a selective inhibitor 5Z-7-Oxozeaenol after MCAO. Neurobehavioral deficits and long-term gray matter and white matter injury were assessed up to 35 days after MCAO. Mi/MΦ functional status and brain inflammatory profiles were assessed 3 days after MCAO by RNA-seq, flow cytometry, and immunohistochemistry. Results- TAK1 Mi/MΦ-specific knockout markedly ameliorated neurological deficits in the rotarod and cylinder tests for at least 35 days after MCAO. Mechanistically, RNA-seq of purified brain Mi/MΦ demonstrated that proinflammatory genes and their predicted biological functions were downregulated or inhibited in microglia and macrophages from TAK1 Mi/MΦ-specific knockout mice versus WT mice 3 days after MCAO. Consistent with the anti-inflammatory phenotype of Mi/MΦ-specific knockout, oxozeaenol treatment mitigated neuroinflammation 3 days after MCAO, manifested by less Iba1+/CD16+ proinflammatory Mi/MΦ and suppressed brain invasion of various peripheral immune cells. Oxozeaenol treatment beginning 2 hours after MCAO improved long-term sensorimotor and cognitive functions in the foot fault, rotarod, and water maze tests. Furthermore, Oxozeaenol promoted both gray matter and white matter integrity 35 days after MCAO. Conclusions- TAK1 promotes ischemia/reperfusion-induced inflammation, brain injury, and maladaptive behavior by enhancing proinflammatory and deleterious Mi/MΦ responses. Therefore, TAK1 inhibition is a promising therapy to improve long-term stroke outcomes.


Assuntos
Comportamento Animal , Lesões Encefálicas/enzimologia , Isquemia Encefálica/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Traumatismo por Reperfusão/enzimologia , Acidente Vascular Cerebral/enzimologia , Animais , Lesões Encefálicas/genética , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , MAP Quinase Quinase Quinases/genética , Macrófagos , Camundongos , Camundongos Knockout , Microglia , Traumatismo por Reperfusão/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Fatores de Tempo , Zearalenona/análogos & derivados , Zearalenona/farmacologia
13.
Am J Physiol Renal Physiol ; 318(4): F878-F887, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32003595

RESUMO

Disruption of mitochondrial dynamics is an important pathogenic event in both acute and chronic kidney diseases, but the underlying mechanism remains poorly understood. Here, we report the regulation of mitofusin-2 (Mfn2; a key mitochondrial fusion protein) by microRNA-214 (miR-214) in renal ischemia-reperfusion that contributes to mitochondrial fragmentation, renal tubular cell death, and ischemic acute kidney injury (AKI). miR-214 was induced, whereas Mfn2 expression was decreased, in mouse ischemic AKI and cultured rat kidney proximal tubular cells (RPTCs) following ATP depletion treatment. Overexpression of miR-214 decreased Mfn2. Conversely, inhibition of miR-214 with anti-miR-214 prevented Mfn2 downregulation in RPTCs following ATP depletion. Anti-miR-214 further ameliorated mitochondrial fragmentation and apoptosis, whereas overexpression of miR-214 increased apoptosis, in ATP-depleted RPTCs. To test regulation in vivo, we established a mouse model with miR-214 specifically deleted from kidney proximal tubular cells (PT-miR-214-/-). Compared with wild-type mice, PT-miR-214-/- mice had less severe tissue damage, fewer apoptotic cells, and better renal function after ischemic AKI. miR-214 induction in ischemic AKI was suppressed in PT-miR-214-/- mice, accompanied by partial preservation of Mfn2 in kidneys. These results unveil the miR-214/Mfn2 axis that contributes to the disruption of mitochondrial dynamics and tubular cell death in ischemic AKI, offering new therapeutic targets.


Assuntos
Lesão Renal Aguda/metabolismo , Apoptose , GTP Fosfo-Hidrolases/metabolismo , Túbulos Renais Proximais/metabolismo , MicroRNAs/metabolismo , Proteínas Mitocondriais/metabolismo , Traumatismo por Reperfusão/metabolismo , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Trifosfato de Adenosina/deficiência , Animais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , GTP Fosfo-Hidrolases/genética , Túbulos Renais Proximais/patologia , Camundongos Knockout , MicroRNAs/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial , Proteínas Mitocondriais/genética , Ratos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
14.
Nat Cell Biol ; 22(2): 225-234, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32029897

RESUMO

Energy stress depletes ATP and induces cell death. Here we identify an unexpected inhibitory role of energy stress on ferroptosis, a form of regulated cell death induced by iron-dependent lipid peroxidation. We found that ferroptotic cell death and lipid peroxidation can be inhibited by treatments that induce or mimic energy stress. Inactivation of AMP-activated protein kinase (AMPK), a sensor of cellular energy status, largely abolishes the protective effects of energy stress on ferroptosis in vitro and on ferroptosis-associated renal ischaemia-reperfusion injury in vivo. Cancer cells with high basal AMPK activation are resistant to ferroptosis and AMPK inactivation sensitizes these cells to ferroptosis. Functional and lipidomic analyses further link AMPK regulation of ferroptosis to AMPK-mediated phosphorylation of acetyl-CoA carboxylase and polyunsaturated fatty acid biosynthesis. Our study demonstrates that energy stress inhibits ferroptosis partly through AMPK and reveals an unexpected coupling between ferroptosis and AMPK-mediated energy-stress signalling.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/genética , Ferroptose/genética , Rim/enzimologia , Peroxidação de Lipídeos/genética , Traumatismo por Reperfusão/genética , Células A549 , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Linhagem Celular Tumoral , Cicloexilaminas/farmacologia , Embrião de Mamíferos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Ácidos Graxos Insaturados/biossíntese , Ferroptose/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glucose/deficiência , Glucose/farmacologia , Humanos , Ferro/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Células MCF-7 , Camundongos , Camundongos Transgênicos , Fenilenodiaminas/farmacologia , Fosforilação , Piperazinas/antagonistas & inibidores , Piperazinas/farmacologia , Cultura Primária de Células , Pirazóis/farmacologia , Pirimidinas/farmacologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética
15.
FASEB J ; 34(2): 2055-2074, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908016

RESUMO

In spinal cord ischemia-reperfusion (I/R) injury, large amounts of reactive oxygen species can cause mitochondrial damage. Therefore, mitophagy acts as the main mechanism for removing damaged mitochondria and protects nerve cells. This study aimed to illustrate the important role of GPCR kinase 2-interacting protein-1 (GIT1) in mitophagy in vivo and in vitro. The level of mitophagy in the neurons of Git1 knockout mice was significantly reduced after ischemia-reperfusion. However, the overexpression of adeno-associated virus with Git1 promoted mitophagy and inhibited the apoptosis of neurons. GIT1 regulated the phosphorylation of Beclin-1 in Thr119, which could promote the translocation of Parkin to the mitochondrial outer membrane. This process was independent of PTEN-induced kinase 1 (PINK1), but it could not rescue the role in the absence of PINK1. Overall, GIT1 enhanced mitophagy and protected neurons against ischemia-reperfusion injury and, hence, might serve as a new research site for the protection of ischemia-reperfusion injury.


Assuntos
Proteína Beclina-1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Mitofagia , Traumatismo por Reperfusão , Doenças da Medula Espinal , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína Beclina-1/genética , Proteínas de Ciclo Celular/genética , Proteínas Ativadoras de GTPase/genética , Camundongos , Camundongos Knockout , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Doenças da Medula Espinal/genética , Doenças da Medula Espinal/metabolismo , Doenças da Medula Espinal/patologia , Doenças da Medula Espinal/prevenção & controle , Ubiquitina-Proteína Ligases/genética
16.
J Immunol ; 204(5): 1322-1333, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31996460

RESUMO

With the development of liver surgery, ischemia-reperfusion (IR) injury has received increasing attention. Roquin-1 has been shown to play an important role in innate immune and immune balance. We demonstrate that Roquin-1 expression increased at 1 h after IR and then decreased in C57B/L mice. The immunofluorescence double-label showed that Roquin-1 was mainly expressed in macrophages (mø). Furthermore, we used clodronate liposomes to remove mø, and injected the bone marrow-derived mø (BMDM) through the tail vein in 1 h before IR. We found that liver IR injury was aggravated by Roquin-1 interference. The results of PCR and ELISA suggested that after interference with Roquin-1, mø increased toward M1 and decreased toward M2. Then, interference with Roquin-1 promoted the polarization of mø to M1 and inhibited the polarization of M2. By Western blot technology and AMPKα and mTOR inhibitors, we found that Roquin-1 promotes the phosphorylation of mTOR and STAT3 by inhibiting the phosphorylation of AMPKα. We used AICAR to activate AMPKα in mø and found that the level of ubiquitination of AMPKα was decreased after activation of AMPKα. Furthermore, by bioinformatics methods, we identified potential ubiquitination sites on AMPKα. By the point mutation experiments in vitro, we confirmed that the ubiquitination of these sites is regulated by Roquin-1. Meanwhile, Roquin-1 interference inhibited the activation and function of AMPKα. This topic describes the protection of liver IR injury by Roquin-1 and discusses its main mechanism for regulating AMPKα activity through ubiquitination and affecting the polarization of mø.


Assuntos
Hepatopatias/imunologia , Fígado/imunologia , Macrófagos/imunologia , Traumatismo por Reperfusão/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/imunologia , Animais , Ativação Enzimática/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Fígado/patologia , Hepatopatias/genética , Hepatopatias/patologia , Macrófagos/patologia , Masculino , Camundongos , Mutação Puntual , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Ubiquitinação/imunologia
17.
Eur J Vasc Endovasc Surg ; 59(1): 98-107, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31744785

RESUMO

OBJECTIVE: This study aimed to investigate the effect of long non-coding RNA (lncRNA) DLGAP1 antisense RNA 1 (DLGAP1-AS1) on vascular endothelial cell (VEC) injury via the phosphoinositide 3-kinase (PI3K)/Akt pathway in rat models of acute lower limb ischaemia-reperfusion (I/R). METHODS: Differentially expressed lncRNAs related to I/R were screened using the gene expression omnibus database. Acute lower limb I/R models were induced in male Wistar rats, in which the regulatory mechanisms of DLGAP1-AS1 silencing were analysed after the treatment of small interfering RNA (siRNA) against DLGAP1-AS1 or an inhibitor of the PI3K/Akt pathway. The relationship between DLGAP1-AS1 and the PI3K/Akt pathway was analysed. The levels of tumour necrosis factor (TNF)-α and vascular cell adhesion molecule-1 (VCAM-1), as well as malondialdehyde (MDA) concentration and creatine kinase (CK) activity, were measured. The number of circulating endothelial cells (CECs) and apoptosis of VECs were identified. RESULTS: Microarray based analysis indicated that DLGAP1-AS1 was highly expressed in I/R, which was further confirmed by detection of expression in rat models of acute lower limb I/R. Notably, the treatment of siRNA against DLGAP1-AS1 led to the activation of the PI3K/Akt pathway. In response to siRNA against DLGAP1-AS1, the levels of TNF-α and VCAM-1 were decreased, and MDA concentration and CK activity was downregulated. Reduced CEC numbers and suppressed VEC apoptosis were also observed. CONCLUSION: DLGAP1-AS1 silencing could further suppress the oxidative stress, exert an anti-apoptosis effect, and reduce inflammatory reaction, whereby VEC injury is alleviated by activation of the PI3K/Akt pathway in rats with acute lower limb I/R.


Assuntos
Apoptose/genética , Células Endoteliais/patologia , RNA Longo não Codificante/metabolismo , Traumatismo por Reperfusão/genética , Transdução de Sinais/genética , Animais , Proliferação de Células/genética , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Masculino , Estresse Oxidativo/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia
18.
Environ Toxicol ; 35(2): 188-193, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31654556

RESUMO

Mitochondrial dynamics and function are important for cell survival regulation under stress. In this study, we report that cerebral ischemia/reperfusion (I/R) injury significantly reduced mitochondrial function through reduced PTEN-induced kinase 1 (PINK1) expression, ATP (Adenosine triphosphate) levels, and increased oxidative stress compared to sham rats. PINK1 overexpression mice significantly improved mitochondrial function by increased mitochondrial complex I, II, and III activities and ATP levels with concomitant decline in reactive oxygen species levels. PINK1 overexpression mice after I/R injury significantly reduced apoptosis through downregulation of cytochrome c, p53 expressions compared to cerebral I/R injury rats. Furthermore, we showed from parkin siRNA studies that PINK1 regulated phosphorylation parkin is critical to the protection against cerebral I/R injury. Altogether, we show that PINK1 mediated parkin regulation is key to the protection against cerebral I/R injury through regulation of mitochondrial function and apoptosis.


Assuntos
Apoptose/genética , Isquemia Encefálica/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases/metabolismo , Traumatismo por Reperfusão/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Sobrevivência Celular/genética , Citocromos c/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estresse Oxidativo/genética , Proteínas Quinases/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
19.
J Ethnopharmacol ; 248: 112319, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31639488

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Apoptosis plays an important role in cerebral ischemia-reperfusion injury and triggers a series of pathological changes which may even be life-threatening. Astragaloside-IV (AS-IV), a natural compound extracted from Astragalus (Astragalus membranaceus (Fisch.) Bunge., Leguminosae, Huangqi in Chinese), showed neuroprotective effects in the study of cerebral ischemia-reperfusion injury. In this study we investigate the effects of AS-IV on apoptosis induced by transient cerebral ischemia and reperfusion in rats, as well as the associated regulatory factors. METHODS: AS-IV was administrated to male Sprague-Dawley (SD) rats after transient cerebral ischemia and reperfusion surgery (12.5, 25, and 50 mg/kg, once per day, continued for 7 days after surgey). After seven days of continuous administration, neurological function, cerebral infarction volume, and pathological changes of brain tissue were detected. Fas, FasL, Caspase-8, Bax, and Bcl-2 mRNA levels were determined by real-time PCR. Caspase-8, Bid, Cytochrome C (Cyto C), cleaved Caspase-3 proteins were determined by western blot and immunohistochemistry was used to quantify Cyto C. RESULTS: AS-IV significantly attenuated the neurological deficit in rats with ischemica-reperfusion injury, and reduced cerebral infarction and neuronal apoptosis. AS-IV inhibited the mRNA upregulation of Fas, FasL, Caspase-8, and Bax/Bcl-2. Furthermore, the protein level of apoptosis cytokines Caspase-8, Bid, cleaved Caspase-3 and Cyto C were also inhibited after ischemia reperfusion, suggesting that AS-IV might alleviate ischemia reperfusion-induced apoptosis by inhibiting the activation of key factors in death receptor pathway and mitochondrial pathway.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Infarto Encefálico/prevenção & controle , Encéfalo/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de Morte Celular/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Encéfalo/metabolismo , Encéfalo/patologia , Infarto Encefálico/genética , Infarto Encefálico/metabolismo , Infarto Encefálico/patologia , Modelos Animais de Doenças , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Ratos Sprague-Dawley , Receptores de Morte Celular/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais
20.
Oxid Med Cell Longev ; 2019: 8189079, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827704

RESUMO

Hepatic ischemia-reperfusion injury (IRI) is a very complex pathological process that is often associated with liver trauma and surgery, especially liver transplantation surgery. Although endoplasmic reticulum stress (ERS) plays a role in this process, the posttranscriptional regulators and the underlying mechanisms are still unclear. Here, we report that the lncRNA AK054386 was increased in hepatic IRI models. Furthermore, AK054386 can act as a "competing endogenous RNA (ceRNA)" and regulate ERS-related factors by binding and sequestering miR-199, which was shown to inhibit ERS in our previous report. Increased expression of AK054386, which might be mediated by activated NF-κB, resulted in sustained ERS and increased cell apoptosis and death in hepatic IRI mouse and cellular models. In contrast, AK054386 inhibition had protective effects on these models. Our data indicate that AK054386 and miR-199 are critical players in hepatic IRI, and we broadened the scope regarding ceRNA mechanisms. We hope that our results will improve the understanding of hepatic IRI and may provide potential therapeutic targets.


Assuntos
Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica , Hepatopatias/patologia , Fígado/irrigação sanguínea , MicroRNAs/genética , RNA Longo não Codificante/genética , Traumatismo por Reperfusão/patologia , Animais , Feminino , Hepatopatias/genética , Hepatopatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais
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