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1.
Yakugaku Zasshi ; 140(8): 1007-1012, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741858

RESUMO

We previously showed that increased permeability of the blood-brain barrier (BBB) after ischemic stroke enables extravasation of nano-sized liposomes and accumulation in the ischemic region, and that delivery of neuroprotective agents using liposomal drug delivery systems (DDS) is applicable for treating cerebral ischemia/reperfusion (I/R) injury. However, entry of liposomes into the brain parenchyma was limited in the early stages after I/R possibly due to microvascular dysfunction induced by pathological progression. As such, new approaches to overcome the BBB are needed. Leukocytes can pass through inflamed BBB in I/R region due to membrane proteins displayed on their surface. We thus hypothesized that incorporation of leukocyte membrane proteins onto liposomal membranes would impart leukocyte-mimicking functions to liposomes and that leukocyte-mimetic liposomes (LM-Lipo) may pass through inflamed endothelial cells and BBB, similar to leukocytes. LM-Lipo prepared using intermembrane protein transfer from human leukemia cells showed significantly increased association to inflamed human umbilical vein endothelial cells relative to plain liposomes. Moreover, LM-Lipo passed through inflamed endothelial cell layer by regulating intercellular junctions. These results suggest that imparting leukocyte-like properties to liposomes via intermembrane protein transfer would be an effective strategy to overcome inflamed endothelial barriers. In this review, we describe our findings on ischemic stroke treatment using liposomal DDS and the potential of LM-Lipo to overcome inflamed endothelial barriers.


Assuntos
Barreira Hematoencefálica/metabolismo , Sistemas de Liberação de Medicamentos , Desenvolvimento de Medicamentos , Leucócitos , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo , Humanos , Permeabilidade , Transporte Proteico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo
2.
Medicine (Baltimore) ; 99(34): e21821, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32846824

RESUMO

BACKGROUND: Traditional Chinese medicine Tongxinluo (TXL) has been widely used to treat coronary artery disease in China, since it could reduce myocardial infarct size and ischemia/reperfusion injury in both non-diabetic and diabetic conditions. It has been shown that TXL could regulate peroxisome proliferator activated receptor-α (PPAR-α), a positive modulator of angiopoietin-like 4 (Angptl4), in diabetic rats. Endothelial junction substructure components, such as VE-cadherin, are involved in the protection of reperfusion injury. Thus, we hypothesized cell-intrinsic and endothelial-specific Angptl4 mediated the protection of TXL on endothelial barrier under high glucose condition against ischemia/reperfusion-injury via PPAR-α pathway. METHODS: Incubated with high glucose medium, the human cardiac microvascular endothelial cells (HCMECs) were then exposed to oxygen-glucose-serum deprivation (2 hours) and restoration (2 hours) stimulation, with or without TXL, insulin, or rhAngptl4 pretreatment. RESULTS: TXL, insulin, and rhAngptl4 had similar protective effects on the endothelial barrier. TXL treatment reversed the endothelial barrier breakdown in HCMECs significantly as identified by decreasing endothelial permeability, upregulating the expression of JAM-A, VE-cadherin, and integrin-α5 and increasing the membrane location of VE-cadherin and integrin-α5, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with small interfering RNA (siRNA) interference and PPAR-α inhibitor MK886 partially abrogated these beneficial effects of TXL. Western blotting also revealed that similar with insulin, TXL upregulated the expression of Angptl4 in HCMECs, which could be inhibited by Angptl4 siRNA or MK886 exposure. TXL treatment increased PPAR-α activity, which could be diminished by MK886 but not by Angptl4 siRNA. CONCLUSION: These data suggest cell-intrinsic and endothelial-specific Angptl4 mediates the protection of TXL against endothelial barrier breakdown during oxygen-glucose-serum deprivation and restoration under high glucose condition partly via the PPAR-α/Angptl4 pathway.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Endotélio/fisiopatologia , PPAR alfa/metabolismo , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/farmacologia , Caderinas/metabolismo , Permeabilidade Capilar , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Vasos Coronários/citologia , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Glucose/farmacologia , Humanos , Indóis/farmacologia , Insulina/farmacologia , Integrina alfa5/metabolismo , Inibidores de Lipoxigenase/farmacologia , Microvasos/citologia , Oxigênio/metabolismo , Oxigênio/farmacologia , Receptores de Superfície Celular/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais
3.
Life Sci ; 257: 118017, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32603821

RESUMO

AIMS: Mesenchymal stem cell (MSC)-derived exosomes (MSCs-exos) regulate biological functions in different diseases, such as liver fibrosis, diabetes, and ischaemic heart injury. However, the function of MSC-derived exosomes on the intestinal barrier and the underlying mechanisms are poorly characterized. MAIN METHODS: The expression of miR-34a/c-5p, miR-29b-3p and Claudin-3 in human normal intestinal tissues and damaged intestinal tissues was evaluated by RT-qPCR. The effect of MSC-secreted exosomes on Claudins in Caco-2 cells was measured by using confocal microscopy, RT-qPCR and Western blot. Dual luciferase reporter assays and RNA immunoprecipitation (RIP) assays were performed to study the interaction between miR-34a/c-5p, miR-29b-3p and Snail. I/R-induced intestinal damage in rats was used to determine the in vivo effect of MSC-exos on intestinal barrier function. KEY FINDINGS: In this study, we found that miR-34a/c-5p, miR-29b-3p and Claudin-3 were downregulated in damaged human intestinal tissues. MSC-exos increased the expression of Claudin-3, Claudin-2 and ZO-1 in Caco-2 cells. Further studies demonstrated that MSC-exos promoted Claudin-3, Claudin-2 and ZO-1 expression in Caco-2 cells by Snail, which was targeted by miR-34a/c-5p and miR-29b-3p. In vivo experiments showed that MSC-derived exosomes could improve I/R-induced intestinal damage through the Snail/Claudins signaling pathway. SIGNIFICANCE: The findings here suggest a novel molecular basis for the therapy of intestinal barrier dysfunction.


Assuntos
Mucosa Intestinal/metabolismo , MicroRNAs/genética , Animais , Condrócitos/metabolismo , Claudinas/metabolismo , Exossomos/genética , Exossomos/metabolismo , Humanos , Intestinos/fisiologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/fisiologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo
4.
Life Sci ; 257: 118004, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32621918

RESUMO

BACKGROUND: Patients undergoing cardiopulmonary bypass (CPB) often develop acute kidney injury (AKI) caused by myocardial ischemia reperfusion (MI/R), and this renal injury can be resolved notably by dexmedetomidine. Endoplasmic reticulum (ER) stress was reported to get involved in organ injury including AKI. OBJECTIVES: The current study aimed to address the correlation between MI/R induced AKI with ER stress and to assess the effects of dexmedetomidine pretreatment on AKI protection. METHOD: Patients selected for heart valve replacement surgery were randomly assigned to NS group (pre-anesthesia with 0.9% NaCl) and DEX group (pre-anesthesia with dexmedetomidine). Rat MI/R model was induced by occluding coronary artery for 30 min followed by 48-hour reperfusion. Rats were randomized into Sham (0.9% NaCl), I/R (MI/R + 0.9% NaCl) and I/R + DEX (MI/R + dexmedetomidine). Organ function and ER stress condition were evaluated by blood chemistry, pathology, and molecular test. RESULTS: Clinical data indicated dexmedetomidine pretreatment attenuated AKI and oxidative stress as well as postischemic myocardial injury in patients. Accordingly animal results suggested dexmedetomidine reduced cellular injury and improved postischemic myocardial and renal function. Dexmedetomidine also reduced myocardial and renal cells apoptosis and down-regulated ER stress. CONCLUSIONS: These results suggested that dexmedetomidine pretreatment attenuates MI/R injury-induced AKI by relieving the ER stress.


Assuntos
Dexmedetomidina/farmacologia , Isquemia Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/prevenção & controle , Idoso , Animais , Apoptose/efeitos dos fármacos , China , Dexmedetomidina/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Feminino , Humanos , Isquemia/metabolismo , Rim/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/metabolismo , Reperfusão Miocárdica/métodos , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estudos Prospectivos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Arterioscler Thromb Vasc Biol ; 40(9): 2279-2292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32611241

RESUMO

OBJECTIVE: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Escherichia coli augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain-containing adapter-inducing interferon-ß) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model. CONCLUSIONS: Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.


Assuntos
Armadilhas Extracelulares/metabolismo , Microbioma Gastrointestinal , Isquemia Mesentérica/metabolismo , Mesentério/irrigação sanguínea , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Traumatismo por Reperfusão/metabolismo , Vênulas/metabolismo , Animais , Bacillus subtilis/patogenicidade , Adesão Celular , Células Cultivadas , Modelos Animais de Doenças , Escherichia coli/patogenicidade , Armadilhas Extracelulares/microbiologia , Feminino , Vida Livre de Germes , Interações Hospedeiro-Patógeno , Migração e Rolagem de Leucócitos , Leucócitos/metabolismo , Leucócitos/microbiologia , Masculino , Isquemia Mesentérica/microbiologia , Isquemia Mesentérica/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/microbiologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Vênulas/microbiologia , Vênulas/patologia
6.
Nat Commun ; 11(1): 3383, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636391

RESUMO

The endogenous repair process can result in recovery after acute kidney injury (AKI) with adaptive proliferation of tubular epithelial cells, but repair can also lead to fibrosis and progressive kidney disease. There is currently limited knowledge about transcriptional regulators regulating these repair programs. Herein we establish the enhancer and super-enhancer landscape after AKI by ChIP-seq in uninjured and repairing kidneys on day two after ischemia reperfusion injury (IRI). We identify key transcription factors including HNF4A, GR, STAT3 and STAT5, which show specific binding at enhancer and super-enhancer sites, revealing enhancer dynamics and transcriptional changes during kidney repair. Loss of bromodomain-containing protein 4 function before IRI leads to impaired recovery after AKI and increased mortality. Our comprehensive analysis of epigenetic changes after kidney injury in vivo has the potential to identify targets for therapeutic intervention. Importantly, our data also call attention to potential caveats involved in use of BET inhibitors in patients at risk for AKI.


Assuntos
Lesão Renal Aguda/genética , Elementos Facilitadores Genéticos , Túbulos Renais/citologia , Lesão Renal Aguda/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Proliferação de Células , Epigênese Genética , Fibrose , Fator 4 Nuclear de Hepatócito/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Nucleares , Receptores de Glucocorticoides/metabolismo , Elementos Reguladores de Transcrição , Traumatismo por Reperfusão/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Fatores de Transcrição , Transcrição Genética
7.
Life Sci ; 256: 117982, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32562693

RESUMO

AIMS: This study was designed to evaluate the protective and therapeutic efficacy of platelet-rich plasma (PRP) against testicular degeneration and germ cell apoptosis after induced spermatic cord torsion/detorsion (TD) in rats. MATERIALS: Forty rats were allocated into 5 groups: 1) control, 2) short torsion/detorsion (STD), 3) long torsion detorsion (LTD), 4) protective (PRP/P) and 5) treatment (PRP/T). Testicular ischemia was induced by twisting the right testis 1080° clockwise for 2.5 h. PRP (10 µl) was injected intra-testicular 5 min before (PRP/P) and 3 h after (PRP/T) detorsion. At the end of the experiment, rats were euthanized at 2, 30, 2, and 30 days for groups 2-5 respectively. Nitric oxide, malondialdehyde, catalase, total antioxidant capacity, reduced glutathione, glutathione-S-transferase, interleukin1 beta, tumor necrosis factor, caspase-3, and B-cell lymphoma 2 expressions were assessed in the testes. Moreover, histological examination was performed. KEY FINDINGS: PRP treatment significantly mitigated the torsion-detorsion induced testicular degeneration. Particularly, by improving the state of oxidative stress (NO, P = 0.0001) and antioxidant markers (TAC, GSH, GST, P = 0.0001-0.01) and decreasing the expression of IL-1ß, TNF-α and cas 3 and increase the BCL2 fold changes (P = 0.0001). The protective use of PRP is superior to the therapeutic use of PRP in the restoration of the testicular histoarchitecture following TD. SIGNIFICANCE: This study illustrates the cyto-protective role of PRP against TD induced testicular cell injury that highlight possible application of PRP as a complementary therapy in different testicular degenerative diseases which might attribute to its ability to ameliorate the oxidative stress and inhibit induced apoptosis.


Assuntos
Plasma Rico em Plaquetas/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Torção do Cordão Espermático/metabolismo , Torção do Cordão Espermático/prevenção & controle , Testículo/metabolismo , Animais , Modelos Animais de Doenças , Masculino , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia , Torção do Cordão Espermático/patologia , Testículo/patologia , Resultado do Tratamento
8.
Life Sci ; 256: 117989, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32565250

RESUMO

AIMS: The beneficial effects of cannabinoid type 2 receptor (CB2R) activation have been verified in various tissue repair processes. Our recent study revealed CB2R activation promotes myogenesis partly through Nrf2 signaling in a mouse skeletal muscle ischemia-reperfusion (IR) injury model. Other relevant mechanisms need to be further elucidated. Macrophages orchestrate tissue regeneration mainly by changing their phenotype and function. The aim of this study was to investigate the role of CB2R in IR-induced skeletal muscle regeneration, focusing on its impact on macrophage polarization and the consequences on myogenesis. MAIN METHODS: The effects of CB2R on skeletal muscle regeneration, and the macrophage infiltration and M1/M2 polarization were tested with the IR injury model in wild type (WT) and CB2R knockout (CB2R-KO) mice. The effect of CB2R on peritoneal macrophage polarization, and its impact on the myoblasts differentiation was evaluated by co-culture experiments in vitro. KEY FINDINGS: The present study revealed the myofiber regeneration was hindered in the CB2R-KO mice. The infiltration of M1 macrophages and relevant markers' protein expression were enhanced in the CB2R-KO mice, while that of M2 macrophages was decreased compared with the WT mice. The in vitro studies further demonstrated that the absence of CB2R promoted M1 polarization while inhibited M2 polarization. The promoted M1 polarization and retarded M2 polarization in CB2R-KO macrophages hindered myoblasts differentiation. SIGNIFICANCE: Overall, these results suggested CB2R plays a beneficial effect on skeletal muscle regeneration partly by regulating macrophage M1/M2 polarization after IR injury in mice.


Assuntos
Polaridade Celular/fisiologia , Macrófagos/fisiologia , Músculo Esquelético/fisiologia , Receptor CB2 de Canabinoide/deficiência , Regeneração/fisiologia , Traumatismo por Reperfusão/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/irrigação sanguínea
9.
Life Sci ; 256: 117987, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32569778

RESUMO

AIMS: Ischemic stroke is the leading cause of severe disability and death worldwide. As the pathogenesis of stroke has not been clearly elucidated and the ability of current therapeutic drugs on crossing the blood-brain barrier (BBB) is extremely low, there is no effective strategy to treat stroke. We aim at investigating the specific advantages of using plasma exosomes (Pla-Exo) for targeting ischemic brain and exploring its underlying mechanism in neuroprotection. MAIN METHODS: Pla-Exo was obtained by a gradient ultracentrifugation of fresh plasma. The quantification of penetrated Pla-Exo through BBB was investigated in vitro BBB model, furthermore, the effects of Pla-Exo and exosomal HSP70 on cerebral ischemia/reperfusion injury were evaluated. KEY FINDINGS: Pla-Exo enhanced BBB crossing by specific interaction between Pla-Exo inherited heat shock protein 70 (HSP70) and endothelial Toll-like receptor 4 (TLR4). As expected, Pla-Exo increased HSP70 expression in the ischemic region through the transfer of HSP70, and led to HSP70 mediated suppression of ROS, thus alleviating cerebral ischemia/reperfusion (I/R) injury by attenuating the deterioration of BBB and preventing mitochondria damage. SIGNIFICANCE: These findings indicated that Pla-Exo can provide protection against ischemia-reperfusion injury via the regulation of HSP70 and it should be further studied as a potential candidate for protection against ischemic injury.


Assuntos
Isquemia Encefálica/metabolismo , Exossomos/metabolismo , Proteínas de Choque Térmico HSP70/administração & dosagem , Plasma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Isquemia Encefálica/prevenção & controle , Endotélio Vascular/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle
10.
Acta Cir Bras ; 35(2): e202000205, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428061

RESUMO

Purpose To investigate the effects of induction of selective liver hypothermia in a rodent model. Methods Seven male Wistar rats were subjected to 90 minutes of partial 70% liver ischemia and topic liver 26°C hypothermia (H group). Other seven male Wistar rats were subjected to 90 minutes of partial 70% normothermic liver ischemia (N group). Five additional rats underwent a midline incision and section of liver ligaments under normothermic conditions and without any liver ischemia (sham group). All animals were sacrificed 24-h after reperfusion, and livers were sampled for analyses. Pathology sections were scored for sinusoidal congestion, ballooning, hepatocelllular necrosis and the presence of neutrophilic infiltrates. Results At the end of the experiment, liver tissue expressions of TNF-ɑ, IL-1ß, iNOS and TNF-ɑ/IL-10 ratio were significantly reduced in the H group compared to N group, whereas IL-10 and eNOS were significantly increased in H group. Histopathological injury scores revealed a significant decrease in ischemia/reperfusion (I/R) injuries in H group. Conclusion Selective liver hypothermia prevented I/R injury by inhibiting the release of inflammatory cytokines, preserves microcirculation, prevents hepatocellular necrosis and leukocyte infiltration, allowing maintenance of the liver architecture.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Hipotermia Induzida/métodos , Fígado/irrigação sanguínea , Traumatismo por Reperfusão/prevenção & controle , Lesão Pulmonar Aguda/patologia , Animais , Temperatura Corporal , Citocinas/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Isquemia/patologia , Fígado/patologia , Masculino , Necrose/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Fator de Necrose Tumoral alfa
11.
Mol Immunol ; 123: 74-87, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32438202

RESUMO

BACKGROUND: Idebenone is a well-appreciated mitochondrial protectant while the mechanisms underlying the neuroprotection in cerebral ischemia and reperfusion (I/R) remain elusive. It has been manifested NLRP3 inflammasom activation contributed to I/R induced damage. It raises questions how exactly NLRP3 inflammasom was activated in microglia and neuron and whether idebenone reverses the process in I/R. METHODS: I/R rat model was utilized and BV2, primary microglia and PC12 cells were subjected to oxygen-glucose deprivation (OGD). Then, western-blotting, q-PCR, immunofluorescence staining, ELISA, flow cytometry and immunoprecipitation analysis were performed. RESULTS: We found ROS-NLRP3 singaling was activated in BV2 cells at OGD/R 24 h. Importantly, microglial NLRP3 activation was essential for NLRP3 activation in PC12 cells under microglial-neuronal co-culture circumstance, which has been confirmed to induced neuronal apoptosis. Further, we found mitochondrial dysfunction in OGD/R led to mt-DNA translocation as well as generation of mt-ROS, resulting cytosolic accumulation of oxidized mt-DNA. Ultimately, oxidized mt-DNA binding to NLRP3 contributed to further activation of NLRP3 and dramatically augmented inflammation in BV2 and PC12 cells. Furthermore, idebenone treatment inhibited the process, thus suppressing the NLRP3-mediated inflammatory injury after OGD/R. In vivo, NLRP3 was activated in microglia of I/R rats and inhibition of NLRP3 was observed in idebenone treatment group, which had less neurological deficit and less infarct volume. INTERPRETATION: Our data revealed the anti-inflammatory effects of idebenone via suppressing activation of NLRP3 and ameliorating NLRP3-mediating damage in I/R, which may provide new insight in therapeutic strategy for ischemic stroke.


Assuntos
Isquemia Encefálica , Encefalite/prevenção & controle , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroproteção/efeitos dos fármacos , Traumatismo por Reperfusão , Ubiquinona/análogos & derivados , Animais , Animais Recém-Nascidos , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/imunologia , Isquemia Encefálica/metabolismo , Células Cultivadas , Encefalite/etiologia , Inflamassomos/metabolismo , Inflamassomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células PC12 , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Ubiquinona/farmacologia , Ubiquinona/uso terapêutico
12.
Life Sci ; 254: 117762, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32437795

RESUMO

AIMS: Patients with nonalcoholic fatty liver disease (NAFLD) have less tolerance to ischemia-reperfusion injury (IRI) of the liver than those with the healthy liver; hence have a higher incidence of severe complications after surgery. This study aimed to investigate the dynamics of the liver and mitochondrial damage and the impact of TLR4 knockout (TLR4KO) on Mfn2 expression in the composite model of NAFLD and IRI. MAIN METHODS: We performed high-fat diet (HFD) feeding and ischemia reperfusion (IR) on wild type (WT) and TLR4 knockout TLR4KO mice. KEY FINDINGS: The degree of structural and functional injuries to the liver and mitochondria (NAFLD and IRI) is greater than that caused by a single factor (NAFLD or IRI) or a simple superposition of both. The IL-6 and TNF-α expressions were significantly suppressed (P < .05), while PGC-1α and Mfn2 expressions were up-regulated considerably (P < .05) after TLR4KO. Furthermore, mitochondrial fusion increased, while ATP consumption and ROS production decreased significantly after TLR4KO (P < .05). The degree of reduction of compound injury by TLR4KO is more significant than the reduction degree of single factor injury. Also, TNF-α and IL-6 levels can be used predictive markers for mitochondrial damage and liver tolerance to NAFLD and IRI. SIGNIFICANCE: TLR4KO upregulates the expression of Mfn2 and PGC-1α in the composite model of NAFLD and IRI. This pathway may be related to IL-6 and TNF-α. This evidence provides theoretical and experimental basis for the subsequent Toll-like receptor 4 (TLR4) receptor targeted therapy.


Assuntos
GTP Fosfo-Hidrolases/biossíntese , Fígado/irrigação sanguínea , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Traumatismo por Reperfusão/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Transplante de Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Reperfusão , Traumatismo por Reperfusão/patologia , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
13.
Life Sci ; 254: 117778, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32407850

RESUMO

Long non-coding RNA (LncRNA) involved in types of physiological insults and diseases via regulating the responses of complex molecular, including cerebral ischemia-reperfusion (I/R) injury. LncRNA SNHG16 played a potential role in ketamine-induced neurotoxicity. In this study, we utilized an in vitro cell model of I/R to examine the specific function and mechanism of LncRNA SNHG16 in oxygen-glucose deprivation and reperfusion (OGD/R) induced SH-SY5Y cells. After in vitro treatment of OGD/R, the lower the SH-SY5Y cell survival, the higher cell the apoptosis and increased caspase-3 activity was observed. Also, OGD/R induced endoplasmic reticulum stress (ERS) through increasing GRP78 and CHOP expressions and down-regulated LncRNA SNHG16 in SH-SY5Y cells. Conversely, LncRNA SNHG16 overexpression promoted OGD/R induced SH-SY5Y cell survival, suppressed its apoptosis, and caspase-3 activity. GRP78 and CHOP expressions were significantly suppressed in LncRNA SNHG16 overexpressing cells. MiR-106b-5p expression was increased and LIMK1 expression was down-regulated in OGD/R induced SH-SY5Y cells, and these effects were reversed by LncRNA SNHG16 overexpression, respectively. Moreover, LIMK1 is a direct target of MiR-106b-5p, and knockdown of LIMK1 reversed the effects of LncRNA SNHG16 on OGD/R-induced SH-SY5Y cells biology. Altogether, these results confirmed an important neuroprotection role of LncRNA SNHG16 in OGD/R induced SH-SY5Y cells injury, and miR-106b-5p/LIMK1 signal axis was involved in the action of LncRNA SNHG16.


Assuntos
Sobrevivência Celular/fisiologia , Quinases Lim/fisiologia , MicroRNAs/fisiologia , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/fisiologia , Traumatismo por Reperfusão/metabolismo , Apoptose/fisiologia , Caspase 3/metabolismo , Células Cultivadas , Regulação para Baixo , Estresse do Retículo Endoplasmático , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico/biossíntese , Humanos , Quinases Lim/genética , Quinases Lim/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/biossíntese
14.
Life Sci ; 254: 117793, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32416164

RESUMO

AIMS: Oxygen and glucose deprivation and reperfusion (OGD/R) injury contributes to the pathophysiology after ischemic stroke, which needs to urgently develop treatment strategies. Previous studies have demonstrated that autophagy in reperfusion period exerted adverse effects on the cerebral ischemic injury. Ginsenoside monomer compound K (CK) is the main intestinal metabolite of ginseng that exerts the pharmacological activities and has a protective effect against cerebral OGD/R injury. However, the specific molecular mechanism of CK protects against OGD/R injury in neurons is still unclear. MATERIALS AND METHODS: In this study, cell viability, reactive oxygen species (ROS) generation, Ca2+ overload, mitochondrial membrane potential depolarization, autophagy and apoptosis were investigated in OGD/R-induced neuronal cells injury after pretreatment with CK and in combination with BML-275 or rapamycin. KEY FINDINGS: Our study found that pretreatment with CK protected neurons against OGD/R injury by increasing cell viability and decreasing the ROS generation, mitochondrial damage, and Ca2+ overload. Moreover, CK cut down autophagy-mediated apoptosis via promoting the process of forming autophagosomes into phagocytic precursors. Furthermore, our study clarified the neuroprotective of CK against OGD/R-induced neural autophagy and apoptosis through the regulation of the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway. SIGNIFICANCE: Taken together, our study provides credible experimental evidence and explains the potential molecular mechanism of CK as one of the main bioactive ingredients of ginseng for the treatment of cerebral ischemia/reperfusion injury.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Morte Celular Autofágica/efeitos dos fármacos , Ginsenosídeos/farmacologia , Glucose/deficiência , Hipóxia/metabolismo , Traumatismo por Reperfusão/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hipóxia/complicações , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Traumatismo por Reperfusão/complicações , Transdução de Sinais/efeitos dos fármacos
15.
PLoS One ; 15(4): e0231978, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315348

RESUMO

Ischemic strokes result in the death of brain tissue and a wave of downstream effects, often leading to lifelong disabilities or death. However, the underlying mechanisms of ischemic damage and repair systems remain largely unknown. In order to better understand these mechanisms, TMT-isobaric mass tagging and mass spectrometry were conducted on brain cortex extracts from mice subjected to one hour of middle cerebral artery occlusion (MCAO) and after one hour of reperfusion. In total, 2,690 proteins were identified and quantified, out of which 65% of the top 5% of up- and down-regulated proteins were found to be significant (p < 0.05). Network-based gene ontology analysis was then utilized to cluster all identified proteins by protein functional groups and cellular roles. Although three different cellular functions were identified-organelle outer membrane proteins, cytosolic ribosome proteins, and spliceosome complex proteins-several functional domains were found to be common. Of these, organelle outer membrane proteins were downregulated whereas cytosolic ribosome and spliceosome complex proteins were upregulated, indicating that major molecular events post-stroke were translation-associated and subsequent signaling pathways (e.g., poly (ADP-ribose) (PAR) dependent cell death). By approaching stroke analyses via TMT-isobaric mass tagging, the work herein presents a grand scope of protein-based molecular mechanisms involved with ischemic stroke recovery.


Assuntos
Córtex Cerebral/metabolismo , Espectrometria de Massas/métodos , Proteoma/metabolismo , Acidente Vascular Cerebral/patologia , Animais , Modelos Animais de Doenças , Regulação para Baixo , Ontologia Genética , Infarto da Artéria Cerebral Média/complicações , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Proteoma/análise , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/genética , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/metabolismo , Regulação para Cima
16.
Zhongguo Zhong Yao Za Zhi ; 45(5): 1142-1148, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32237458

RESUMO

Serum metabonomic profiles of the model of focal cerebral ischemia reperfusion is established with the suture-occluded method by Longa to study the effect of ginsenosides. In this study, 48 rats were randomly divided into six groups: sham-operated group, pathological model group, positive drug group(6 mg·kg~(-1)·d~(-1)) and high, medium, low-dose ginsenosides groups(200, 100, 50 mg·kg~(-1)·d~(-1)). They are given intragastric administration respectively with same amount of 0.5% CMC-Na,nimodipine and ginsenoside for 5 days. At 2 h after the final administration, the model was established with the suture-occluded method, and free radical-scavenging activity changes of ginsenoside were observed by maillard reaction, and Longa was possible used as a renoprotective agent-occluded method. At the end of 24 h after the reperfusion, the hemolymph of rats in each group was collected, and the ~1H-NMR spectrum was collected after being treated by certain methods, and analyzed by principal component analysis(PCA). Compared with sham-operated group, pathological model group showed significant increases in the levels of lactate, glutamate, taurine, choline, glucose and methionine, but decreases in the levels of 3-hydroxybutyrate and phosphocreatine/creatine in serum. After treatment with ginsenosides, lipid, 3-hydroxybutyrate and phosphocreatine/creatine were increased in the serum of ginsenosides group rats, but with decreases in lactate and glutamate. The results showed that ginsenosides could regulate metabolic disorders in rats with focal cerebral ischemia reperfusion, and promote a recovery in the process of metabolism. It's helpful to promote the metabolic changes in rats with focal cerebral ischemia reperfusion via ~1H-NMR, and lay a foundation to develop ginsenosides as a new drug to treat ischemic cerebral paralysis.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Ginsenosídeos/farmacologia , Metaboloma , Traumatismo por Reperfusão/tratamento farmacológico , Ácido 3-Hidroxibutírico , Animais , Isquemia Encefálica/metabolismo , Creatina , Hemolinfa , Fosfocreatina , Espectroscopia de Prótons por Ressonância Magnética , Distribuição Aleatória , Ratos , Traumatismo por Reperfusão/metabolismo
17.
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
18.
Invest Ophthalmol Vis Sci ; 61(3): 51, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32232350

RESUMO

Purpose: The lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages. Methods: We examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells. Results: LysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons. Conclusions: LysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.


Assuntos
Antígenos CD/metabolismo , Encéfalo/metabolismo , Caderinas/metabolismo , Integrases/metabolismo , Substâncias Luminescentes/metabolismo , Proteínas Luminescentes/metabolismo , Muramidase/metabolismo , Vasos Retinianos/metabolismo , Animais , Animais Recém-Nascidos , Pesquisa Biomédica , Diagnóstico por Imagem , Endotélio Vascular/metabolismo , Feminino , Genes Reporter , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Neurônios/metabolismo , Recombinação Genética , Traumatismo por Reperfusão/metabolismo , Células Ganglionares da Retina/metabolismo
19.
Basic Res Cardiol ; 115(3): 26, 2020 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-32146560

RESUMO

Mesenchymal stromal cells (MSCs) exhibit antiapoptotic and proangiogenic functions in models of myocardial infarction which may be mediated by secreted small extracellular vesicles (sEVs). However, MSCs have frequently been harvested from aged or diseased patients, while the isolated sEVs often contain high levels of impurities. Here, we studied the cardioprotective and proangiogenic activities of size-exclusion chromatography-purified sEVs secreted from human foetal amniotic fluid stem cells (SS-hAFSCs), possessing superior functional potential to that of adult MSCs. We demonstrated for the first time that highly pure (up to 1.7 × 1010 particles/µg protein) and thoroughly characterised SS-hAFSC sEVs protect rat hearts from ischaemia-reperfusion injury in vivo when administered intravenously prior to reperfusion (38 ± 9% infarct size reduction, p < 0.05). SS-hAFSC sEVs did not protect isolated primary cardiomyocytes in models of simulated ischaemia-reperfusion injury in vitro, indicative of indirect cardioprotective effects. SS-hAFSC sEVs were not proangiogenic in vitro, although they markedly stimulated endothelial cell migration. Additionally, sEVs were entirely responsible for the promigratory effects of the medium conditioned by SS-hAFSC. Mechanistically, sEV-induced chemotaxis involved phosphatidylinositol 3-kinase (PI3K) signalling, as its pharmacological inhibition in treated endothelial cells reduced migration by 54 ± 7% (p < 0.001). Together, these data indicate that SS-hAFSC sEVs have multifactorial beneficial effects in a myocardial infarction setting.


Assuntos
Líquido Amniótico/citologia , Cardiotônicos/metabolismo , Movimento Celular , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Quimiotaxia , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Ratos , Traumatismo por Reperfusão/patologia
20.
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
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