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1.
Invest Ophthalmol Vis Sci ; 61(1): 3, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31995154

RESUMO

Purpose: The purpose of this study was to investigate the expression of death ligands in the lacrimal glands (LGs), identify upstream factors that regulate their expression, and determine the functional roles of these factors in the pathogenesis of dry eye disease (DED). Methods: For DED experiment, ex vivo coculture system with LG and in vivo murine model using a controlled environment chamber were utilized. C57BL/6 mice and hypoxia-inducible factor (HIF)-1α conditional knockout (CKO) mice were used. Immunohistochemical staining, polymerase chain reaction, and immunoblotting were performed to determine levels of death ligands including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DED-induced LGs. Additionally, acinar cell and CD45+ cell apoptosis was determined with neutralizing TRAIL treatment. Results: Desiccating stress significantly increased HIF-1α expression in LG-acinar cells. Furthermore, HIF-1α deficiency significantly enhanced the infiltration of CD45+ inflammatory cells in LG and induced LG-acinar cell death. Meanwhile, only TRAIL expression was increased in DED-LG, but abrogated in HIF-1α CKO. Interestingly, the main source of TRAIL was the CD45- LG-acinar cells, but not CD45+ immune cells after DED induction. Using ex vivo coculture system, we confirmed LG-induced apoptosis of immune cells via HIF-1α-mediated TRAIL secretion following DED. Consistent with ex vivo, the insufficiency of HIF-1α and TRAIL enhanced recruitment of inflammatory cells to the LG and subsequently exacerbated ocular surface damage in DED mice. Conclusions: Our findings offer novel insight into the regulatory function of acinar cell-derived TRAIL in limiting inflammatory damage and could be implicated in the development of potential therapeutic strategies for DED.


Assuntos
Dacriocistite/metabolismo , Síndromes do Olho Seco/metabolismo , Regulação da Expressão Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Aparelho Lacrimal/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose , Técnicas de Cocultura , Dacriocistite/patologia , Modelos Animais de Doenças , Síndromes do Olho Seco/patologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Aparelho Lacrimal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real
2.
Acta Cir Bras ; 34(11): e201901102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31859816

RESUMO

PURPOSE: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. METHODS: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. RESULTS: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). CONCLUSION: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.


Assuntos
Apoptose/efeitos dos fármacos , Cinamatos/farmacologia , Glucosídeos Iridoides/farmacologia , Óxido Nítrico/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Testículo/irrigação sanguínea , Animais , Western Blotting , Heme Oxigenase-1/análise , Marcação In Situ das Extremidades Cortadas , Masculino , Malondialdeído/análise , NADP/análise , Peroxidase/análise , Distribuição Aleatória , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Reprodutibilidade dos Testes , Superóxido Dismutase/análise , Testículo/patologia , Xantina Oxidase/análise
3.
Invest Ophthalmol Vis Sci ; 60(14): 4849-4857, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747684

RESUMO

Purpose: We reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain. Methods: C57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice. Results: Anisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice. Conclusions: Our study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.


Assuntos
Biossíntese de Proteínas/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anisomicina/farmacologia , Proteínas de Ciclo Celular/genética , Morte Celular , Eletrorretinografia , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica/fisiologia , Marcação In Situ das Extremidades Cortadas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parvovirinae/genética , Inibidores da Síntese de Proteínas/farmacologia , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Transfecção
4.
Invest Ophthalmol Vis Sci ; 60(14): 4670-4680, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31725166

RESUMO

Purpose: Long noncoding RNAs (lncRNAs) are important in disease progression and cellular functions. This study aimed to conduct global lncRNA profiling and characterize the role of lncRNA 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta 3-sence RNA 1 (PLCD3-OT1) in the progression of age-related cataract (ARC). Methods: We performed lncRNA expression profiling of lens capsule from ARC groups and age-matched groups using high-throughput RNA-sequencing. Real-time PCR was conducted to detect the expression pattern of lncRNA and mRNA in the clinical samples and cell model. Assays of cell-counting kit-8, 5'-ethynyl-2'-deoxyuridine, TUNEL, and propidium iodide staining were used to detect cell viability, proliferation, apoptosis, and cell cycle. We also performed fluorescence in situ hybridization assay to detect the location of lncRNA, and verified the endogenous competitive RNA mechanism between miRNAs, lncRNAs, and target genes via double-luciferase reporter analyses. Results: The expression of lncRNA PLCD3-OT1 and PLCD3 were significantly decreased in ARC. PLCD3-OT1 overexpression promoted the expression of PLCD3, cell viability, proliferation, and inhibited cell apoptosis upon oxidative stress, while knockdown of PLCD3 showed the opposite results. Mechanistically, PLCD3-OT1functions through positively regulation the expression of PLCD3. In addition, PLCD3-OT1 may act as a ceRNA to regulate the expression of PLCD3 through competition for miR-224-5p. Conclusions: PLCD3-OT1 and PLCD3 may become potential therapeutic targets for the prognosis, diagnosis, and treatment of ARC.


Assuntos
Catarata/prevenção & controle , MicroRNAs/metabolismo , Fosfolipase C delta/fisiologia , RNA Longo não Codificante/fisiologia , Idoso , Western Blotting , Catarata/metabolismo , Catarata/patologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Marcação In Situ das Extremidades Cortadas , Lentivirus/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Transfecção
5.
Invest Ophthalmol Vis Sci ; 60(14): 4759-4773, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31738824

RESUMO

Purpose: Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods: Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results: After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions: This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.


Assuntos
Diferenciação Celular/fisiologia , Colinérgicos/farmacologia , Células Ependimogliais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/embriologia , Transdução de Sinais/fisiologia , Esferoides Celulares/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Embrião de Galinha , Colina O-Acetiltransferase/metabolismo , Crioultramicrotomia , Ácido Glutâmico/farmacologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neurônios Retinianos/citologia , Esferoides Celulares/metabolismo , Fixação de Tecidos , Vimentina/metabolismo
6.
Invest Ophthalmol Vis Sci ; 60(13): 4360-4377, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31634394

RESUMO

Purpose: To investigate the neuroprotective properties of creatine in the retina using in vitro and in vivo models of injury. Methods: Two different rat retinal culture systems (one containing retinal ganglion cells [RGC] and one not) were subjected to either metabolic stress, via treatments with the mitochondrial complex IV inhibitor sodium azide, or excitotoxic stress, via treatment with N-methyl-D-aspartate for 24 hours, in the presence or absence of creatine (0.5, 1.0, and 5.0 mM). Neuronal survival was assessed by immunolabeling for cell-specific antigens. Putative mechanisms of creatine action were investigated in vitro. Expression of creatine kinase (CK) isoenzymes in the rat retina was examined using Western blotting and immunohistochemistry. The effect of oral creatine supplementation (2%, wt/wt) on retinal and blood creatine levels was determined as well as RGC survival in rats treated with N-methyl-D-aspartate (NMDA; 10 nmol) or high IOP-induced ischemia reperfusion. Results: Creatine significantly prevented neuronal death induced by sodium azide and NMDA in both culture systems. Creatine administration did not alter cellular adenosine triphosphate (ATP). Inhibition of CK blocked the protective effect of creatine. Retinal neurons, including RGCs, expressed predominantly mitochondrial CK isoforms, while glial cells expressed exclusively cytoplasmic CKs. In vivo, NMDA and ischemia reperfusion caused substantial loss of RGCs. Creatine supplementation led to elevated blood and retinal levels of this compound but did not significantly augment RGC survival in either model. Conclusions: Creatine increased neuronal survival in retinal cultures; however, no significant protection of RGCs was evident in vivo, despite elevated levels of this compound being present in the retina after oral supplementation.


Assuntos
Creatina/farmacologia , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Células Cultivadas , Creatina Quinase/metabolismo , Eletrorretinografia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Isoenzimas/metabolismo , N-Metilaspartato/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Retina/enzimologia , Retina/fisiopatologia , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Azida Sódica/farmacologia , Estresse Fisiológico
7.
Invest Ophthalmol Vis Sci ; 60(13): 4346-4359, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31626691

RESUMO

Purpose: Glaucoma is a neurodegenerative eye disease characterized by gradually impaired visual field and irreversible blindness due to retinal ganglion cell (RGC) loss. Our previous studies have confirmed that hydrogen sulfide (H2S) takes part in the glaucomatous process and contributes to RGC protection. The present study aimed to further investigate the role of extracellular signal-regulated kinase 1/2 (ERK 1/2) pathway underlying the impact of H2S, to better understand the mechanism through which H2S exerts neuroprotection in glaucoma. Methods: An established rat glaucoma model was used and 168 rats were qualified to undergo sodium hydrosulfide (NaHS, a H2S donor)/PD98059 (an ERK inhibitor) treatment. Then the survival and apoptosis of RGC were evaluated through retrograde labeling and TUNEL staining, along with activity evaluations of ERK 1/2 pathway, intrinsic apoptotic pathway, glial activation, nuclear factor kappa B (NF-κB) pathway, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, autophagy, and TNF-α production through immunohistochemistry, Western blotting, and ELISA. Results: The study demonstrated that NaHS suppressed ERK 1/2 pathway activity similarly to PD98059 in retinas of experimental glaucoma rats, while PD98059 also similarly suppressed glial activation, NF-κB pathway, NADPH oxidase, and TNF-α production. However, PD98059 did not affect RGC survival, apoptotic regulation, or autophagy as NaHS did. Conclusions: Our study indicated that inhibition of ERK 1/2 pathway might partly contribute to the neuroprotection by H2S in experimental glaucoma; however, it was insufficient to initiate the therapeutic effect on its own.


Assuntos
Glaucoma/prevenção & controle , Sulfeto de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Autofagia , Western Blotting , Sobrevivência Celular/fisiologia , Flavonoides/farmacologia , Marcação In Situ das Extremidades Cortadas , Pressão Intraocular/fisiologia , Masculino , Modelos Animais , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Neuroproteção , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/patologia , Fator de Necrose Tumoral alfa/metabolismo
8.
Invest Ophthalmol Vis Sci ; 60(12): 3776-3785, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31503282

RESUMO

Purpose: To investigate the therapeutic effects of targeting signal transducer and activator of transcription-3 (STAT3) activation on the ocular surface damage of dry eye in mice. Methods: Adult Balb/C and C57BL/6 mice with benzalkonium chloride (BAC) treatment, lacrimal gland excision, and meibomian gland dysfunction were used as dry eye models. The levels of phosphorylated STAT3 (p-STAT3) were detected with immunofluorescence staining and Western blotting. STAT3 inhibition was performed by topical application of STAT3 inhibitor S3I-201. Corneal epithelial barrier function, tear production, and conjunctival goblet cell density were quantified with fluorescein sodium staining, phenol red cotton test, and histochemical staining. The expressions of matrix metalloproteinase (MMP)-3/9, TUNEL, and inflammation cytokines were assessed with immunofluorescence staining, qPCR, and ELISA assays. The therapeutic effect of S3I-201 was further compared with the Janus kinase inhibitor tofacitinib and ruxolitinib. Results: Elevated levels of nuclear p-STAT3 were detected in the corneal and conjunctival epithelium of three dry eye models. Topical application of S3I-201 improved corneal epithelial barrier function, increased tear production and conjunctival goblet cell density in BAC-induced dry eye mice. Moreover, S3I-201 decreased the expression of MMP-3/9, suppressed the apoptosis of corneal and conjunctival epithelial cells, and reduced the levels of IL-1ß, IL-6, IL-17A, and IFN-γ. Compared with tofacitinib and ruxolitinib, the STAT3 inhibitor S3I-201 showed superior improvement of tear production and inflammatory cytokine expression in lacrimal gland. Conclusions: Elevated STAT3 activation is involved in the pathogenesis of dry eye, while targeting STAT3 effectively alleviates BAC-induced ocular surface damage.


Assuntos
Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Proteínas Inibidoras de STAT Ativados/uso terapêutico , Fator de Transcrição STAT3/antagonistas & inibidores , Administração Oftálmica , Animais , Western Blotting , Túnica Conjuntiva/metabolismo , Citocinas/metabolismo , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Ensaio de Imunoadsorção Enzimática , Epitélio/metabolismo , Epitélio Anterior/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Células Caliciformes/patologia , Marcação In Situ das Extremidades Cortadas , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Soluções Oftálmicas , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/metabolismo , Lágrimas/fisiologia
9.
Invest Ophthalmol Vis Sci ; 60(10): 3538-3546, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415077

RESUMO

Purpose: To determine if trigeminal innervations of the corneal epithelium maintains its integrity and homeostasis through controlling the nicotinamide adenine dinucleotide (NAD) content of this tissue. Methods: Corneal denervation of C57BL/6 mice was induced by squeezing the nerve bundles that derive from the trigeminal ganglion and was confirmed by whole-mount corneal nerve staining and the sensation test. The apoptosis of the corneal epithelium was examined by TUNEL assay and annexin V/propidium iodide staining. NAD biosynthesis-related enzymes were analyzed by quantitative PCR, immunofluorescence staining, and Western blotting. FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), exogenous nicotinamide mononucleotide (NMN), and NAD+ were used to evaluate the effect of NAD+ on the apoptosis of cultured corneal epithelial cells and epithelial detachment in denervated mice. Protein expression that related to apoptosis and phosphorylation were analyzed by Western blotting. Results: The denervated mice showed spontaneous corneal epithelial detachment and cell apoptosis accompanied with impaired epithelial NAD+ contents due to low levels of NAMPT. Similarly, inhibition of NAMPT recapitulated epithelial detachment as in denervated mice and induced apoptosis in cultured corneal epithelial cells. The replenishment of NMN or NAD+ partially slowed down corneal nerve fiber degeneration, reduced the epithelial defect in denervated mice, and improved apoptosis induction in FK866-treated cells by restoring the activation levels of SIRT1, AKT, and CREB. Conclusions: Corneal denervation lowered epithelial NAD+ contents through reducing the expression of NAMPT and caused cell apoptosis and epithelial defects, suggesting that corneal innervations contribute to epithelial homeostasis by regulating NAD+ biosynthesis.


Assuntos
Apoptose , Córnea/inervação , Denervação , Epitélio Anterior/patologia , NAD/metabolismo , Nervo Trigêmeo/fisiologia , Animais , Anexina A5/metabolismo , Western Blotting , Proteína de Ligação a CREB/metabolismo , Córnea/metabolismo , Doenças da Córnea/diagnóstico , Doenças da Córnea/metabolismo , Epitélio Anterior/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo , Doenças do Nervo Trigêmeo/diagnóstico , Doenças do Nervo Trigêmeo/metabolismo , Proteína X Associada a bcl-2/metabolismo
10.
Invest Ophthalmol Vis Sci ; 60(10): 3481-3491, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408114

RESUMO

Purpose: This study investigated the therapeutic effects of puerarin (PR) on a rat model of anterior ischemic optic neuropathy (rAION). Methods: The neuroprotective effects of PR on rAION were evaluated using flash visual-evoked potentials (FVEP), retrograde labeling of retinal ganglion cells (RGCs), TUNEL assay of the retina, optical coherence tomography (OCT) images of optic nerve width, and ED1 staining of the optic nerve (ON). The inflammatory response of ON and Akt signaling pathways were analyzed through Western blot. M2 polarization was determined by immunostaining and immunoblotting in ONs. Results: In FVEP analysis, the amplitude of P1-N2 and the RGC density in the PR-treated group were 2.3- and 1.6-fold higher than those in the PBS-treated group, respectively (P < 0.05). The number of apoptotic RGC in the PR-treated group was 2.8-fold lower than that in the PBS-treated group. OCT images demonstrated that PR treatment-reduced ON edema in the acute phase compared to PBS treatment (P < 0.05). Macrophage infiltration was reduced by 5.2-fold by PR treatment compared with the PBS treatment (P < 0.05). PR treatment inhibited the levels of iNOS, IL-1ß, and TNF-α, induced the levels of IL-10, Arg1, and Fizz1 in the rAION model. The levels of p-Akt1 and C/EBPß in the PR-treated group increased by 3.4-fold and 5.89-fold compared with those in the PBS-treated group (P < 0.05). Inhibition of Akt activation reduced the number of M2 macrophage in the PR-treated group (P < 0.05). Conclusions: PR treatment provided the neuroprotective effects in the rAION model, which may lead to new clinical applications.


Assuntos
Anti-Inflamatórios/uso terapêutico , Apoptose/efeitos dos fármacos , Isoflavonas/uso terapêutico , Neuropatia Óptica Isquêmica/tratamento farmacológico , Vasodilatadores/uso terapêutico , Animais , Western Blotting , Cromonas/administração & dosagem , Cromonas/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Potenciais Evocados Visuais/fisiologia , Marcação In Situ das Extremidades Cortadas , Injeções Intravítreas , Masculino , Morfolinas/administração & dosagem , Morfolinas/farmacologia , Neuropatia Óptica Isquêmica/diagnóstico por imagem , Neuropatia Óptica Isquêmica/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Células Ganglionares da Retina/patologia , Tomografia de Coerência Óptica
11.
Pediatr Surg Int ; 35(11): 1309-1316, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31435735

RESUMO

PURPOSE: Undescended testes (UDT) are subjected to heat stress, which can disturb gonocyte transformation as well as apoptosis. This study aims to describe the apoptosis pathway occurring during minipuberty of children with unilateral (UDT), and to investigate the role of inhibin-B. METHODS: Testicular biopsies at unilateral orchidopexy of 10 boys (6-9 months old) with normal inhibin-B (n = 5) or low inhibin-B (n = 5) were selected for immunohistochemistry and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labelling) assay. Testicular tubules were labelled with antibodies against Anti-Müllerian hormone (AMH, Sertoli cell marker), mouse Vasa Homolog (MVH) and placental alkaline phosphatase (PLAP) (both germ cell markers), cleaved caspase3 (apoptotic marker), and followed by confocal imaging and cell counting with Fiji/ImageJ. Data were analyzed with GraphPad Prism. RESULTS: In males with low and normal inhibin-B, there was no statistical difference (p > 0.05) in the percentage of testicular tubules containing TUNEL + cells, number of cleaved caspase3 ± germ cells/tubule, total number of germ cells/tubule, and the percentage of fibrotic tubules or number of Sertoli cells/tubule. CONCLUSIONS: These results suggest that inhibin-B does not regulate cell death of gonocytes and further studies are required to uncover any role of inhibin-B in gonocyte transformation.


Assuntos
Diferenciação Celular , Criptorquidismo/patologia , Inibinas/sangue , Túbulos Seminíferos/citologia , Apoptose , Caspase 3/metabolismo , Criptorquidismo/cirurgia , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Lactente , Masculino , Orquidopexia
12.
Invest Ophthalmol Vis Sci ; 60(10): 3264-3274, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31369671

RESUMO

Purpose: Lacrimal gland excision (LGE) has been utilized in several studies to model aqueous tear deficiency, yet sex as a biological variable has not been factored in to these reports. This study compared corneal pathology in male and female mice following LGE-induced dry eye. Methods: An LGE of either the extraorbital lacrimal gland (single LGE) or both the extraorbital and intraorbital lacrimal glands (double LGE) was performed in male and female C57BL/6J and Balb/cJ mice to produce dry eye of graded severity. Following excision, tearing was evaluated with phenol red thread, and corneal fluorescein staining was scored to quantify the severity of damage. Corneas were evaluated for apoptosis by the TUNEL assay and for cell proliferation using Ki67 staining. Furthermore, corneas were harvested and analyzed for macrophages via flow cytometry. Results: Baseline tearing levels were similar in male and female mice, and LGE resulted in comparable reductions in tearing with the lowest levels recorded after double LGE. As determined by fluorescein staining, LGE produced more severe damage to the cornea in female C57BL/6J and Balb/cJ mice. Double LGE increased TUNEL and Ki67 staining in the cornea, with greater increases found in female mice. Furthermore, LGE produced a greater increase in the total number of corneal macrophages in female mice. Conclusions: These results indicate that female mice are more susceptible to LGE-induced corneal damage. The mechanisms involved in producing these sex differences still need to be elucidated but may involve increased inflammation and macrophage infiltration.


Assuntos
Doenças da Córnea/fisiopatologia , Síndromes do Olho Seco/fisiopatologia , Aparelho Lacrimal/cirurgia , Animais , Doenças da Córnea/etiologia , Doenças da Córnea/metabolismo , Síndromes do Olho Seco/etiologia , Síndromes do Olho Seco/metabolismo , Feminino , Citometria de Fluxo , Fluoresceína/metabolismo , Corantes Fluorescentes/metabolismo , Marcação In Situ das Extremidades Cortadas , Inflamação/patologia , Antígeno Ki-67/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fatores Sexuais , Lágrimas/fisiologia
13.
Invest Ophthalmol Vis Sci ; 60(10): 3669-3679, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31469894

RESUMO

Purpose: To investigate the presence and role of fibroblast senescence in the dynamic process of corneal wound healing involving stromal cell apoptosis, proliferation, and differentiation. Methods: An in vivo corneal wound healing model was performed using epithelial debridement in C57BL/6 mice. The corneas were stained using TUNEL, Ki67, and α-smooth muscle actin (α-SMA) as markers of apoptosis, proliferation, and myofibroblastic differentiation, respectively. Cellular senescence was confirmed by senescence-associated ß-galactosidase (SA-ß-gal) staining and P16Ink4a expression. Mitogenic response and gene expression were compared among normal fibroblasts, H2O2-induced senescent fibroblasts, and TGF-ß-induced myofibroblasts in vitro. The senescence was further detected in mouse models of corneal scarring, alkali burn, and penetrating keratoplasty (PKP). Results: The apoptosis and proliferation of corneal stromal cells were found to peak at 4 and 24 hours after epithelial debridement. Positive staining of SA-ß-gal was observed clearly in the anterior stromal cells at 3 to 5 days. The senescent cells displayed P16Ink4a+ vimentin+ α-SMA+, representing the major origin of activated corneal resident fibroblasts. Compared with normal fibroblasts and TGF-ß-induced myofibroblasts, H2O2-induced senescent fibroblasts showed a nonfibrogenic phenotype, including a reduced response to growth factor basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB), increased matrix metalloproteinase (MMP)1/3/13 expression, and decreased fibronectin and collagen I expression. Moreover, cellular senescence was commonly found in the mouse corneal scarring, alkali burn, and PKP models. Conclusions: Corneal epithelial debridement induced the senescence of corneal fibroblasts after apoptosis and proliferation. The senescent cells displayed a nonfibrogenic phenotype and may be involved in the self-limitation of corneal fibrosis.


Assuntos
Senescência Celular/fisiologia , Lesões da Córnea/fisiopatologia , Fibroblastos/citologia , Cicatrização/fisiologia , Actinas/metabolismo , Animais , Apoptose , Western Blotting , Proliferação de Células , Lesões da Córnea/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Peróxido de Hidrogênio/toxicidade , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Hidróxido de Sódio/toxicidade
14.
Nutrients ; 11(7)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31331066

RESUMO

Vasicinone is a quinazoline alkaloid isolated from the Adhatoda vasica plant. In this study, we explored the neuroprotective effect and underlying molecular mechanism of vasicinone against paraquat-induced cellular apoptosis in SH-SY5Y cells. Vasicinone reduced the paraquat-induced loss of cell viability, rescued terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL)-positive apoptotic nuclei, and suppressed generation of reactive oxygen species (ROS) in a dose-dependent manner. Western blotting analysis revealed that vasicinone increased the phosphorylation of IGF1R/PI3K/AKT cell survival signaling molecules and downregulated the paraquat-induced, mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinase (JNK)-mediated apoptotic pathways compared to that observed in cells not treated with vasicinone. This protection depended critically on the activation of IGF1R, and the silencing of IGF1R by siRNA completely abrogated the protective effect of vasicinone in SH-SY5Y cells. Our findings indicated that vasicinone is a potential candidate for the treatment of Parkinson's disease and possibly other oxidative stress-related neurodegenerative disorders.


Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Paraquat/farmacologia , Doença de Parkinson , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Marcação In Situ das Extremidades Cortadas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
15.
Pharmacology ; 104(3-4): 196-206, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31336368

RESUMO

AIM: The present study was performed to investigate the effect of Aesculus hippocastanum (AH; Venoplant®) on concanavalin A (ConA)-induced acute liver injury and explore the mechanism in mice. METHODS: ConA (20 mg/kg) was administered via tail vein injection to induce hepatic damage. The groups of AH (Venoplant®) were given at 65.8, 131.6, and 263.2 mg/kg by oral gavages for 20 days. The serum levels of aspartate transaminase (AST), alanine aminotransferase (ALT), total protein (TP), and albumin (Alb) were determined by automatic biochemical analyzer, and the Alb/globulin (A/G) ratio was calculated. Tumor necrosis factor-α (TNF-α) and IFN-γ levels were assayed by enzyme-linked immunosorbent assay. The liver tissue was attained by hematoxylin and eosin, and the histopathological changes were calculated. The cell apoptosis was assayed by terminal dUTP nick-end labeling. The malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) content of liver tissue were assayed by related kits. The activity of caspase-3 was detected by spectrophotometry. The expressions of cytochrome c, Bax, Bcl-2, c-Jun N-terminal kinase (JNK), and p-JNK were detected by western blot. RESULTS: The results showed that the levels of ALT, AST, IFN-γ, and TNF-α in AH (Venoplant®) groups were significantly lower than those in ConA-injured group, while the levels of TP, Alb, and A/G were significantly higher. The SOD and GSH levels were significantly increased, and the MDA level was decreased; liver histopathology was changed consistently with the serological indicators, AH (Venoplant®) treatment significantly reduced the pathological damage and cell apoptosis; while in AH (Venoplant®) group, the expressions of cytochrome c, caspase-3, Bax/Bcl-2 ratio, and p-JNK were significantly decreased. CONCLUSION: AH (Venoplant®) could significantly protect the ConA-induced acute liver injury in mice via inhibition of reactive oxygen species and JNK pathway.


Assuntos
Aesculus/química , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Concanavalina A/farmacologia , Substâncias Protetoras/farmacologia , Alanina Transaminase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/metabolismo , Caspase 3/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Glutationa/metabolismo , Marcação In Situ das Extremidades Cortadas/métodos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
16.
Mediators Inflamm ; 2019: 7329131, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31263382

RESUMO

Apoptosis of CD4+ T cells plays a central role in the progression of sepsis because it is associated with subsequent immunosuppression and the lack of specific treatment. Thus, developing therapeutic strategies to attenuate the apoptosis of CD4+ T cells in sepsis is critical. Several studies have demonstrated that Mdivi-1, which is a selective inhibitor of the dynamin-related protein 1 (Drp1), attenuates apoptosis of myocardial cells and neurons during various pathologic states. The present study revealed the impact of Mdivi-1 on the apoptosis of CD4+ T cells in sepsis and the potential underlying mechanisms. We used lipopolysaccharide (LPS) stimulation and cecal ligation and puncture (CLP) surgery as sepsis models in vitro and in vivo, respectively. Our results showed that Mdivi-1 attenuated the apoptosis of CD4+ T cells both in vitro and in vivo. The potential mechanism underlying the protective effect of Mdivi-1 involved Mdivi-1 reestablishing mitochondrial fusion-fission balance in sepsis, as reflected by the expression of the mitofusin 2 (MFN2) and optic atrophy 1 (OPA1) , Drp1 translocation, and mitochondrial morphology, as observed by electron microscopy. Moreover, Mdivi-1 treatment reduced reactive oxygen species (ROS) production and prevented the induction of endoplasmic reticulum stress (ERS) and associated apoptosis. After using tunicamycin to activate ER stress, the protective effect of Mdivi-1 on CD4+ T cells was reversed. Our results suggested that Mdivi-1 ameliorated apoptosis in CD4+ T cells by reestablishing mitochondrial fusion-fission balance and preventing the induction of endoplasmic reticulum stress in experimental sepsis.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Quinazolinonas/uso terapêutico , Sepse/tratamento farmacológico , Sepse/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Atrofia Óptica Autossômica Dominante/metabolismo , Espécies Reativas de Oxigênio/metabolismo
17.
Int Heart J ; 60(4): 944-957, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31257341

RESUMO

Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.


Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/genética , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/patologia
18.
ACS Appl Mater Interfaces ; 11(33): 29536-29548, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31333014

RESUMO

Previous reports from our team revealed the significant potential advantage of Mn-Zn ferrite nanoparticles (NPs) in magnetic resonance imaging (MRI), whereas anisotropic NPs reportedly increased the blood circulation time of nanocarriers. Thus, anisotropic Mn-Zn ferrite displayed a huge potential in cancer synchronous diagnosis and treatment, that is, enhanced MRI observation was performed simultaneously when drug-targeted delivery therapy was applied to the tumor. Here, we developed three shaped Mn-Zn ferrite (Mn0.63Zn0.37Fe2O4) MNPs used as cancer theranostic nanoagents and compared the effect of the three shaped MNPs on cancer theranostics. Compared to the monodisperse sphere MNPs (S-MNPs-PPR) and clustering MNPs (C-MNPs-PPR), worm-like Mn-Zn ferrite MNPs (W-MNPs-PPR) achieved better results in T2-weighted MRI and achieved more sustained drug release than S-MNPs-PPR and more complete drug release than C-MNPs-PPR in vitro. Additionally, polyethylene glycol (PEG) coating and RGD modification encouraged the three shaped MNPs to evade the recruitment of macrophages more easily and to target the integrin-enriched endothelial cells instead. Meanwhile, W-MNPs-PPR coupled with Paclitaxel (PTX) exhibited more delivery of PTX in the integrin-enriched cells than the other two shaped MNPs, and the content of PTX was far more than that of the wild-type Taxol control group. What is more, in vivo results demonstrated that PTX-coated W-MNPs-PPR not only gained good dual-mode imaging in the tumor (MRI and fluorescence images) but also achieved longer blood circulation time and more PTX-targeted delivery to the tumor, as well as more efficiency in tumor cell killing, which make the simultaneous diagnosis and treatment of tumors to be conducted. Therefore, our works further revealed the importance of the NP shape on its functionality and ultimately provided an alternative and efficient worm-like theranostic nanoagent for tumor theranostics.


Assuntos
Compostos Férricos/química , Manganês/química , Nanopartículas/química , Nanomedicina Teranóstica/métodos , Zinco/química , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Marcação In Situ das Extremidades Cortadas , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Paclitaxel/química , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Polietilenoglicóis/química , Células RAW 264.7 , Espectroscopia de Luz Próxima ao Infravermelho
19.
BMC Ophthalmol ; 19(1): 146, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291924

RESUMO

BACKGROUND: Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis (CMV). Our previous results have shown that there is a functional relationship between autophagy and apoptosis during MCMV infection of retinal pigment epithelium (RPE). The purpose of this study was to determine whether autophagy plays a significant role in the death of retinal cells during MCMV retinitis. METHODS: The retinas of adult BALB/c mice were infected with MCMV via supraciliary injection. Rapamycin, a mTOR inhibitor, was injected to MCMV-infected BALB/c mice intraperitoneally. Immunohistochemistry and western blot were performed to observe the spread pattern of virus in retinas and the levels of targeted proteins. Plaque assay was performed to determine the virus titer in different groups. Since Atg5 is a key gene regulating autophagy, we bred Atg5flox/flox; Nestin-Cre mice to deeply elucidate the role of autophagy during MCMV retinitis. Atg5flox/flox; Nestin-Cre mice were genotyped and infected with MCMV. Immunohistochemistry was performed to observe the type of virus-infected cells and apoptosis in retinas during MCMV retinitis. RESULTS: In MCMV mouse model, MCMV infection in outer nuclear layer (ONL) and inner nuclear layer (INL) in the retinas caused cleaved caspase 3 positive apoptosis, which is not co-localized with early antigen (EA) positive virus infected cells in rapamycin treated group. Rapamycin treatment increased the levels of LC3B-II by inhibiting mTOR and decreased the levels of cleaved caspase-3 during MCMV retinitis. However, virus propagation was not affected by rapamycin. In Atg5flox/flox; Nestin-Cre mice, RPE and glial cells were the main targets of viral infection, and number of EA positive retinal cells and TUNEL positive retinal cells was significantly increased compared to Atg5flox/+; Nestin-Cre mice though there was no difference of virus propagation between Atg5flox/flox; Nestin-Cre mice and Atg5flox/+; Nestin-Cre mice. CONCLUSIONS: Autophagy protects retinal cells from MCMV infection induced apoptosis through mTOR-mediated signaling pathway.


Assuntos
Apoptose , Retinite por Citomegalovirus/patologia , Infecções Oculares Virais/patologia , Epitélio Pigmentado da Retina/patologia , Animais , Autofagia , Western Blotting , Morte Celular , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C
20.
Oxid Med Cell Longev ; 2019: 4579806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191799

RESUMO

Acute myocardial infarction (AMI) is the leading cause of sudden death worldwide. MicroRNA-155 (miR-155) has been reported to target antiapoptotic genes in various diseases models, but the functional role of miR-155 in response to MI injury needs further investigations. This study investigated the role of miR-155 in myocardial ischemia injury. TUNEL and flow cytometry were performed to measure cell apoptosis. Western blot analysis was employed to detect protein expressions of Bcl-2, XIAP, Bax, and caspase-3. qRT-PCR was used to quantify miRNA levels. We showed that miR-155 was dynamically elevated in murine hearts subjected to MI and in neonatal rat ventricular cardiomyocyte (NRVM) injury induced by hydrogen peroxide (H2O2). In response to H2O2, the silencing of miR-155 using AMO-155 (antisense inhibitor oligodeoxyribonucleotides) significantly increased cell viability and reduced cell apoptosis. Moreover, AMO-155 reversed the H2O2-induced downregulation of Bcl-2 and XIAP and upregulation of Bax and cleaved-caspase-3. Further study revealed that AMO-155 resulted in a decrease of H2O2-induced JC-1-labelled monomeric cell number. In addition, AMO-155 markedly decreased infarct size, ameliorated impaired cardiac function, and significantly reduced apoptotic cell percentages in MI mice heart. The RNA-binding protein Quaking (QKI) was predicted as a target gene of miR-155 through bioinformatic analysis, and AMO-155 attenuated the downregulation of QKI in H2O2-treated cardiomyocytes and MI mice heart. Knockdown of QKI by siRNA abolished the antiapoptotic effects of AMO-155. Taken together, miR-155 is upregulated in the MI heart and NRVMs in response to H2O2 stress, and downregulating of miR-155 protects cardiomyocytes against apoptosis. Mechanistically, it is probably due to the repression of QKI signaling pathway.


Assuntos
MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Células Cultivadas , Peróxido de Hidrogênio/farmacologia , Marcação In Situ das Extremidades Cortadas , Masculino , MicroRNAs/genética , Infarto do Miocárdio/genética , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
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