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1.
Biomaterials ; 313: 122753, 2025 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-39217793

RESUMO

Non-viral nanoparticles (NPs) have seen heightened interest as a delivery method for a variety of clinically relevant nucleic acid cargoes in recent years. While much of the focus has been on lipid NPs, non-lipid NPs, including polymeric NPs, have the possibility of improved efficacy, safety, and targeting, especially to non-liver organs following systemic administration. A safe and effective systemic approach for intracellular delivery to the lungs could overcome limitations to intratracheal/intranasal delivery of NPs and improve clinical benefit for a range of diseases including cystic fibrosis. Here, engineered biodegradable poly (beta-amino ester) (PBAE) NPs are shown to facilitate efficient delivery of mRNA to primary human airway epithelial cells from both healthy donors and individuals with cystic fibrosis. Optimized NP formulations made with differentially endcapped PBAEs and systemically administered in vivo lead to high expression of mRNA within the lungs in BALB/c and C57 B/L mice without requiring a complex targeting ligand. High levels of mRNA-based gene editing were achieved in an Ai9 mouse model across bronchial, epithelial, and endothelial cell populations. No toxicity was observed either acutely or over time, including after multiple systemic administrations of the NPs. The non-lipid biodegradable PBAE NPs demonstrate high levels of transfection in both primary human airway epithelial cells and in vivo editing of lung cell types that are targets for numerous life-limiting diseases particularly single gene disorders such as cystic fibrosis and surfactant deficiencies.


Assuntos
Pulmão , Camundongos Endogâmicos C57BL , Nanopartículas , Polímeros , RNA Mensageiro , Animais , Pulmão/metabolismo , Humanos , Nanopartículas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Polímeros/química , Camundongos Endogâmicos BALB C , Camundongos , Fibrose Cística , Feminino , Ligantes , Células Epiteliais/metabolismo
2.
Int J Biol Sci ; 20(11): 4476-4495, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39247815

RESUMO

Chronic stress is closely associated with gastrointestinal disorders. However, the impact of stress-related neurotransmitters such as serotonin (5-hydroxytryptamine, 5-HT) on the intestines under chronic stress conditions remains poorly understood. This study aims to elucidate the mechanisms by which 5-HT affects mitochondrial biogenesis and intestinal barrier integrity during chronic stress. Employing a chronic restraint stress (CRS) mouse model, we observed elevated intestinal 5-HT levels, altered colonic mucosal structure, and disrupted tight junctions. The increase in 5-HT was associated with up-regulated serotonin synthesis enzymes and downregulated serotonin reuptake transporters, indicating an imbalance in serotonin homeostasis imbalance caused by chronic stress. Furthermore, serotonin exacerbated oxidative stress and impaired tight junction protein expression, highlighting its role in promoting intestinal barrier dysfunction. Experiments with cells in vitro demonstrated that 5-HT impairs mitochondrial biogenesis by inhibiting the AMPK-PGC-1α axis via 5-HT7 receptors and the cAMP-PKA pathway. Pharmacological inhibition of serotonin synthesis or 5-HT7 receptors alleviated the intestinal barrier damage caused by 5-HT and chronic stress, restoring mitochondrial biogenesis. These findings provide compelling evidence that serotonin exacerbates chronic stress-induced intestinal barrier disruption by inhibiting the AMPK-PGC-1α axis, paving the way for novel therapeutic interventions targeting the detrimental effects of serotonin on the intestine, particularly under chronic stress conditions.


Assuntos
Mitocôndrias , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Serotonina , Serotonina/metabolismo , Animais , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Mitocôndrias/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Proteínas Quinases Ativadas por AMP/metabolismo , Camundongos Endogâmicos C57BL
3.
Theranostics ; 14(12): 4773-4786, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39239521

RESUMO

Gene therapy using a protein-based CRISPR system in the brain has practical limitations due to current delivery systems, especially in the presence of arterial occlusion. To overcome these obstacles and improve stability, we designed a system for intranasal administration of gene therapy for the treatment of ischemic stroke. Methods: Nanoparticles containing the protein-based CRISPR/dCas9 system targeting Sirt1 were delivered intranasally to the brain in a mouse model of ischemic stroke. The CRISPR/dCas9 system was encapsulated with calcium phosphate (CaP) nanoparticles to prevent them from being degraded. They were then conjugated with ß-hydroxybutyrates (bHb) to target monocarboxylic acid transporter 1 (MCT1) in nasal epithelial cells to facilitate their transfer into the brain. Results: Human nasal epithelial cells were shown to uptake and transfer nanoparticles to human brain endothelial cells with high efficiency in vitro. The intranasal administration of the dCas9/CaP/PEI-PEG-bHb nanoparticles in mice effectively upregulated the target gene, Sirt1, in the brain, decreased cerebral edema and increased survival after permanent middle cerebral artery occlusion. Additionally, we observed no significant in vivo toxicity associated with intranasal administration of the nanoparticles, highlighting the safety of this approach. Conclusion: This study demonstrates that the proposed protein-based CRISPR-dCas9 system targeting neuroprotective genes in general, and SIRT1 in particular, can be a potential novel therapy for acute ischemic stroke.


Assuntos
Administração Intranasal , Encéfalo , Modelos Animais de Doenças , Terapia Genética , AVC Isquêmico , Nanopartículas , Sirtuína 1 , Animais , Camundongos , Humanos , AVC Isquêmico/terapia , AVC Isquêmico/genética , Nanopartículas/administração & dosagem , Terapia Genética/métodos , Sirtuína 1/genética , Sirtuína 1/metabolismo , Encéfalo/metabolismo , Masculino , Fosfatos de Cálcio , Sistemas CRISPR-Cas , Camundongos Endogâmicos C57BL , Células Endoteliais/metabolismo , Isquemia Encefálica/terapia , Isquemia Encefálica/genética , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/genética , Células Epiteliais/metabolismo
4.
PLoS One ; 19(9): e0310001, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39240898

RESUMO

Obstructive uropathy is a common kidney disease caused by elevated hydrostatic pressure (HP), but relevant molecular and cellular mechanisms have not yet been well understood. In this study, we ex vivo investigated the effects of elevated HP on human renal epithelial cells (HREpCs). Primary HREpCs were subjected to 100 cmH2O HP for 8 or 48 h. Then, the cells were cultured without HP stimulation for another 24 h or 72 h. Cell morphology showed almost no change after 8h HP treatment, but exhibited reversible elongation after 48h HP treatment. HP treatment for 8 h increased the expression of TGFB1 and VEGFA but decreased the expression of CSF2 and TGFB2. On the other hand, HP treatment for 48 h downregulated the expression of CSF2, TGFB2, PDGFB, VEGFA, and VEGFB, while upregulated the expression of TGFB3. Interestingly, all changes induced by 48 h HP treatment were detected more severe compared to 8 h HP treatment. In conclusion, elongated ex vivo HP loading to renal epithelial cells induces reversible changes on cell morphology and disturbs the expression of several growth factors, which provides novel mechanistic insight on elevated HP-caused kidney injury such as obstructive uropathy.


Assuntos
Células Epiteliais , Pressão Hidrostática , Rim , Humanos , Células Epiteliais/metabolismo , Rim/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Transformador beta1/metabolismo
5.
Ecotoxicol Environ Saf ; 283: 116985, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-39217894

RESUMO

Cigarette smoke, a complex mixture produced by tobacco combustion, contains a variety of carcinogens and can trigger DNA damage. Overactivation of c-MET, a receptor tyrosine kinase, may cause cancer and cellular DNA damage, but the underlying mechanisms are unknown. In this work, we investigated the mechanisms of cigarette smoke extract (CSE) induced malignant transformation and DNA damage in human bronchial epithelial cells (BEAS-2B). The results demonstrated that CSE treatment led to up-regulated mRNA expression of genes associated with the c-MET signaling pathway, increased expression of the DNA damage sensor protein γ-H2AX, and uncontrolled proliferation in BEAS-2B cells. ATR, ATR, and CHK2, which are involved in DNA damage repair, as well as the phosphorylation of c-MET and a group of kinases (ATM, ATR, CHK1, CHK2) involved in the DNA damage response were all activated by CSE. In addition, CSE activation promotes the phosphorylation modification of ATR, CHK1 proteins associated with DNA damage repair. The addition of PHA665752, a specific inhibitor of c-MET, or knock-down with c-MET both attenuated DNA damage, while overexpression of c-MET exacerbated DNA damage. Thus, c-MET phosphorylation may be involved in CSE-induced DNA damage, providing a potential target for intervention in the prevention and treatment of smoking-induced lung diseases.


Assuntos
Brônquios , Dano ao DNA , Células Epiteliais , Nicotiana , Proteínas Proto-Oncogênicas c-met , Fumaça , Humanos , Proteínas Proto-Oncogênicas c-met/metabolismo , Fosforilação/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Brônquios/citologia , Fumaça/efeitos adversos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Linhagem Celular , Transdução de Sinais/efeitos dos fármacos , Produtos do Tabaco
6.
Sci Rep ; 14(1): 20409, 2024 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-39223207

RESUMO

Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and was the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while the exons encoding p14ARF remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina , Células Epiteliais , Próstata , Neoplasias da Próstata , Telomerase , Humanos , Masculino , Telomerase/genética , Telomerase/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Linhagem Celular
7.
Cell Mol Life Sci ; 81(1): 385, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-39235496

RESUMO

Cisplatin-induced renal tubular injury largely restricts the wide-spread usage of cisplatin in the treatment of malignancies. Identifying the key signaling pathways that regulate cisplatin-induced renal tubular injury is thus clinically important. PARVB, a focal adhesion protein, plays a crucial role in tumorigenesis. However, the function of PARVB in kidney disease is largely unknown. To investigate whether and how PARVB contributes to cisplatin-induced renal tubular injury, a mouse model (PARVB cKO) was generated in which PARVB gene was specifically deleted from proximal tubular epithelial cells using the Cre-LoxP system. In this study, we found depletion of PARVB in proximal tubular epithelial cells significantly attenuates cisplatin-induced renal tubular injury, including tubular cell death and inflammation. Mechanistically, PARVB associates with transforming growth factor-ß-activated kinase 1 (TAK1), a central regulator of cell survival and inflammation that is critically involved in mediating cisplatin-induced renal tubular injury. Depletion of PARVB promotes cisplatin-induced TAK1 degradation, inhibits TAK1 downstream signaling, and ultimately alleviates cisplatin-induced tubular cell damage. Restoration of PARVB or TAK1 in PARVB-deficient cells aggravates cisplatin-induced tubular cell injury. Finally, we demonstrated that PARVB regulates TAK1 protein expression through an E3 ligase ITCH-dependent pathway. PARVB prevents ITCH association with TAK1 to block its ubiquitination. Our study reveals that PARVB deficiency protects against cisplatin-induced tubular injury through regulation of TAK1 signaling and indicates targeting this pathway may provide a novel therapeutic strategy to alleviate cisplatin-induced kidney damage.


Assuntos
Cisplatino , MAP Quinase Quinase Quinases , Camundongos Knockout , Transdução de Sinais , Cisplatino/efeitos adversos , Cisplatino/toxicidade , Animais , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Camundongos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Antineoplásicos/farmacologia , Antineoplásicos/efeitos adversos , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal
8.
Mol Med Rep ; 30(4)2024 10.
Artigo em Inglês | MEDLINE | ID: mdl-39219265

RESUMO

Indole­3­propionic acid (IPA), a product of Clostridium sporogenes metabolism, has been shown to improve intestinal barrier function. In the present study, in vitro experiments using NCM460 human colonic epithelial cells were performed to investigate how IPA alleviates lipopolysaccharide (LPS)­induced intestinal epithelial cell injury, with the aim of improving intestinal barrier function. In addition, the underlying mechanism was explored. NCM460 cell viability and apoptosis were measured using the Cell Counting Kit­8 assay and flow cytometry, respectively. The integrity of the intestinal epithelial barrier was evaluated by measuring transepithelial electrical resistance (TEER). The underlying molecular mechanism was explored using western blotting, immunofluorescence staining, a dual luciferase reporter gene assay and quantitative PCR. The results showed that 10 µg/ml LPS induced the most prominent decrease in cell viability after 24 h of treatment. By contrast, IPA effectively inhibited LPS­induced apoptosis in the intestinal epithelial cells. Additionally, >0.5 mM IPA improved intestinal barrier function by increasing TEER and upregulating the expression of tight junction proteins (zonula occludens­1, claudin­1 and occludin). Furthermore, IPA inhibited the release of pro­inflammatory cytokines (IL­1ß, IL­6 and TNF­α) in a dose­dependent manner and this was achieved via regulation of the Toll­like receptor 4 (TLR4)/myeloid differentiation factor 88/NF­κB and TLR4/TRIF/NF­κB pathways. In conclusion, IPA may alleviate LPS­induced inflammatory injury in human colonic epithelial cells. Taken together, these results suggest that IPA may be a potential therapeutic approach for the management of diseases characterized by LPS­induced intestinal epithelial cell injury and intestinal barrier dysfunction.


Assuntos
Apoptose , Células Epiteliais , Indóis , Mucosa Intestinal , Lipopolissacarídeos , NF-kappa B , Transdução de Sinais , Receptor 4 Toll-Like , Humanos , Receptor 4 Toll-Like/metabolismo , NF-kappa B/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Indóis/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Função da Barreira Intestinal
9.
Front Immunol ; 15: 1441908, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39224597

RESUMO

Introduction: The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route. Methods: Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rß that constitute the bovIFN-λ3 receptor. Results: Our immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rß subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rß subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells. Conclusion: Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells.


Assuntos
Interferon lambda , Interferons , Conchas Nasais , Animais , Bovinos , Interferons/metabolismo , Interferons/imunologia , Conchas Nasais/virologia , Conchas Nasais/imunologia , Conchas Nasais/metabolismo , Antivirais/farmacologia , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/fisiologia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Células Epiteliais/virologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Interleucinas/genética , Interleucinas/farmacologia , Interleucinas/imunologia , Interleucinas/metabolismo , Linhagem Celular , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Proteínas Recombinantes/farmacologia , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/metabolismo , Receptores de Citocinas
10.
Anim Biotechnol ; 35(1): 2391520, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39222080

RESUMO

MicroRNAs (miRNAs) were identified to be involved in various biological functions by regulating the degradation or suppressing the translation of their downstream target genes. Recent studies have identified miR-29a play a key role in functions of mammal cell differentiation, proliferation, apoptosis, and signal transduction. However, the underlying functions for miR-29a in jejunal epithelial cells biological function still to be investigated. In order to explore the yak jejunal epithelial cells proliferation and barrier dysfunction with over expression of miR-29a gene, three 0-day-old Pamir male yaks were randomly selected and slaughtered in present study, and the jejunal epithelial cells were isolated and cultured to determine yak jejunal epithelial cells proliferation and protein composition on differential expression of miR-29a gene in Pamir plateau. Here, we demonstrated that the overexpression of miR-29a gene could inhibit the proliferation of Pamir yaks jejunum epithelial cells, and contribute to the apoptosis of Pamir yaks jejunal epithelial cells with some extent. A total of 133 differentially expressed proteins were identified in different expression of miR-29a groups by label-free Mass Spectrometry (MS), which could be concluded to two predominant themes: cell proliferation and inflammatory response. Interestingly, GPR41, as a bridge protein, was contacted two predominant themes to involved in Pamir Yaks jejunal mechanical barrier PPI network, and the target proteins displayed strong mutual interactions in the complex PPI network. Overall, our study suggested that the over-expression miR-29a inhibited the jejunal epithelial cells proliferation and the expressions of specific proteins, which damaged jejunal barrier function to slow down the intestine structure and function advanced mature development during young livestock period for influence the enhanced performance of production efficiency.


Assuntos
Apoptose , Proliferação de Células , Células Epiteliais , Jejuno , MicroRNAs , Animais , Bovinos/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Células Epiteliais/fisiologia , Células Epiteliais/metabolismo , Apoptose/genética , Apoptose/fisiologia , Jejuno/citologia , Jejuno/metabolismo , Proliferação de Células/genética , Masculino
11.
PLoS One ; 19(9): e0310253, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39283878

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that can cause severe bacterial pneumonia. Amygdalin is the main active pharmaceutical ingredient of bitter almond, which has broad-spectrum antibacterial, anti-inflammatory, anti-oxidation and immunomodulatory effects. It is also the main ingredient of Yinhua Pinggan granule, which is commonly used to moisten the lung and relieve cough. However, little is known about the effects of amygdalin on MRSA. In this study, we found that amygdalin exhibited good antimicrobial activity in vitro against MRSA. Amygdalin has a protective effect on MRSA infected cells, and the effect is better when combined with levofloxacin. It also can reduce the adhesion and invasion of MRSA to cells. Amygdalin has anti-inflammatory and antioxidant effects, which can significantly reduce the increase of inflammatory factors and the production of ROS caused by infection. The protective mechanism of amygdalin on cells may be related to inhibiting the expression of NLRP3, ASC and IL-1ß pyroptosis pathways. Taken together, our study suggests that amygdalin exerts antibacterial effects by affecting biofilm formation, the expression of virulence factors, and drug resistance genes. Amygdalin combined with levofloxacin has a protective effect on A549 cells infected with MRSA, and the mechanism may be related to the inhibition of inflammatory response, oxidative damage and pyroptosis.


Assuntos
Amigdalina , Antibacterianos , Inflamação , Staphylococcus aureus Resistente à Meticilina , Estresse Oxidativo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Amigdalina/farmacologia , Humanos , Estresse Oxidativo/efeitos dos fármacos , Células A549 , Antibacterianos/farmacologia , Inflamação/tratamento farmacológico , Inflamação/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Biofilmes/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Levofloxacino/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
12.
Nat Commun ; 15(1): 7900, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39261485

RESUMO

Semen quality and fertility has declined over the last 50 years, corresponding to ever-increasing environmental stressors. However, the cellular mechanisms involved and their impact on sperm functions remain unknown. In a repeated sampling human cohort study, we identify a significant effect of prior perceived stress to increase sperm motility 2-3 months following stress, timing that expands upon our previous studies revealing significant stress-associated changes in sperm RNA important for fertility. We mechanistically examine this post-stress timing in mice using an in vitro stress model in the epididymal epithelial cells responsible for sperm maturation and find 7282 differentially H3K27me3 bound DNA regions involving genes critical for mitochondrial and metabolic pathways. Further, prior stress exposure significantly changes the composition and size of epithelial cell-secreted extracellular vesicles that when incubated with mouse sperm, increase mitochondrial respiration and sperm motility, adding to our prior work showing impacts on embryo development. Together, these studies identify a time-dependent, translational signaling pathway that communicates stress experience to sperm, ultimately affecting reproductive functions.


Assuntos
Mitocôndrias , Motilidade dos Espermatozoides , Espermatozoides , Animais , Masculino , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Humanos , Camundongos , Mitocôndrias/metabolismo , Respiração Celular , Vesículas Extracelulares/metabolismo , Epididimo/metabolismo , Camundongos Endogâmicos C57BL , Estresse Fisiológico , Adulto , Células Epiteliais/metabolismo , Análise do Sêmen , Estudos de Coortes
13.
Physiol Rep ; 12(17): e70021, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39261977

RESUMO

Many pathological conditions lead to defects in intestinal epithelial integrity and loss of barrier function; Sphingosine-1-phosphate (S1P) has been shown to augment intestinal barrier integrity, though the exact mechanisms are not completely understood. We have previously shown that overexpression of Sphingosine Kinase 1 (SphK1), the rate limiting enzyme for S1P synthesis, significantly increased S1P production and cell proliferation. Here we show that microRNA 495 (miR-495) upregulation led to decreased levels of SphK1 resultant from a direct effect at the SphK1 mRNA. Increasing expression of miR-495 in intestinal epithelial cells resulted in decreased proliferation and increased susceptibility to apoptosis. Transgenic expression of miR-495 inhibited mucosal growth, as well as decreased proliferation in the crypts. The intestinal villi also expressed decreased levels of barrier proteins and exaggerated damage upon exposure to cecal ligation-puncture. These results implicate miR-495 as a critical negative regulator of intestinal epithelial protection and proliferation through direct regulation of SphK1, the rate limiting enzyme critical for production of S1P.


Assuntos
Apoptose , Mucosa Intestinal , Lisofosfolipídeos , MicroRNAs , Fosfotransferases (Aceptor do Grupo Álcool) , Esfingosina , MicroRNAs/metabolismo , MicroRNAs/genética , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Animais , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Mucosa Intestinal/metabolismo , Camundongos , Proliferação de Células , Regulação para Baixo , Células Epiteliais/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
16.
Cell Mol Life Sci ; 81(1): 404, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-39277835

RESUMO

Proliferation of renal tubular epithelial cells (TEC) is essential for restoring tubular integrity and thereby to support renal functional recovery from kidney ischemia/reperfusion (KI/R) injury. Activation of transcriptional factor c-Myc promotes TEC proliferation following KI/R; however, the mechanism regarding c-Myc activation in TEC is incompletely known. Heat shock protein A12A (HSPA12A) is an atypic member of HSP70 family. In this study, we found that KI/R decreased HSPA12A expression in mouse kidneys and TEC, while ablation of HSPA12A in mice impaired TEC proliferation and renal functional recovery following KI/R. Gain-of-functional studies demonstrated that HSPA12A promoted TEC proliferation upon hypoxia/reoxygenation (H/R) through directly interacting with c-Myc and enhancing its nuclear localization to upregulate expression of its target genes related to TEC proliferation. Notably, c-Myc was lactylated in TEC after H/R, and this lactylation was enhanced by HSPA12A overexpression. Importantly, inhibition of c-Myc lactylation attenuated the HSPA12A-induced increases of c-Myc nuclear localization, proliferation-related gene expression, and TEC proliferation. Further experiments revealed that HSPA12A promoted c-Myc lactylation via increasing the glycolysis-derived lactate generation in a Hif1α-dependent manner. The results unraveled a role of HSPA12A in promoting TEC proliferation and facilitating renal recovery following KI/R, and this role of HSPA12A was achieved through increasing lactylation-mediated c-Myc activation. Therefore, targeting HSPA12A in TEC might be a viable strategy to promote renal functional recovery from KI/R injury in patients.


Assuntos
Proliferação de Células , Células Epiteliais , Proteínas de Choque Térmico HSP70 , Túbulos Renais , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-myc , Traumatismo por Reperfusão , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Camundongos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Humanos , Rim/metabolismo , Rim/patologia
17.
J Transl Med ; 22(1): 844, 2024 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-39285426

RESUMO

BACKGROUND: Ocular toxicity is a severe adverse effect that limits the chronic clinical use of the antiarrhythmic drug amiodarone. Here, we aimed to evaluate the cytoprotective effect of artemisinin and explore the potential signalling pathways in human retinal pigment epithelial (RPE) cell cultures. METHODS: D407 cell cultures were exposed to amiodarone and the impact of artemisinin was evaluated. The key parameters included lactate dehydrogenase (LDH) release, intracellular reactive oxygen species (ROS) generation, and the mitochondrial membrane potential (MMP). We also assessed the protein levels of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), phosphorylated adenosine monophosphate-activated protein kinase (AMPK)ɑ (p-AMPK), calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), and nuclear factor erythroid 2-related factor 2 (Nrf2). RESULTS: Artemisinin reduced the cytotoxicity induced by amiodarone, as reflected by decreased LDH release, ROS generation, and MMP disruption. Additionally, artemisinin increased p-AMPK, CaMKK2, and Nrf2 protein levels. Inhibition of AMPK, CaMKK2, or Nrf2 abolished the cytoprotective effect of artemisinin. AMPK activation and Nrf2 knockdown further supported its protective role. CONCLUSIONS: Artemisinin protected RPE cells from amiodarone-induced damage via the CaMKK2/AMPK/Nrf2 pathway. The in vivo experiments in mice confirmed its efficacy in preventing retinal injury caused by amiodarone. These results suggest that an artemisinin-based eye formulation could be repurposed for treating amiodarone-induced ocular toxicity.


Assuntos
Proteínas Quinases Ativadas por AMP , Amiodarona , Artemisininas , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Citoproteção , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Espécies Reativas de Oxigênio , Epitélio Pigmentado da Retina , Transdução de Sinais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Humanos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Amiodarona/efeitos adversos , Amiodarona/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Artemisininas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Camundongos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia
18.
BMC Vet Res ; 20(1): 390, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39227948

RESUMO

BACKGROUND: This study aimed to identify the roles of L-tryptophan (Trp) and its rate-limiting enzymes on the receptivity of bovine endometrial epithelial cells. Real-time PCR was conducted to analyze the differential expression of genes between different groups of bovine endometrial epithelial cells. Western blot was performed to detect Cyclooxygenase-2 (COX2) expression after treatment with Trp or kynurenine (the main metabolites of Trp). The kynurenine assay was used to examine if Trp or prostaglandin E2 (PGE2) can increase the production of kynurenine in the bovine endometrial epithelial cells. RESULTS: Trp significantly stimulates insulin growth factor binding protein 1 (IGFBP1) expression, a common endometrial marker of conceptus elongation and uterus receptivity for ruminants. When bovine endometrial epithelial cells are treated with Trp, tryptophan hydroxylase-1 remains unchanged, but tryptophan 2,3-dioxygenase 2 (TDO2) is significantly increased, suggesting tryptophan is mainly metabolized through the kynurenine pathway. Kynurenine significantly stimulates IGFBP1 expression. Furthermore, Trp and kynurenine significantly increase the expression of aryl hydrocarbon receptor (AHR). CH223191, an AHR inhibitor, abrogates the induction of Trp and kynurenine on IGFBP1. PGE2 significantly induces the expression of TDO2, AHR, and IGFBP1. CONCLUSIONS: The regulation between Trp / kynurenine and PGE2 may be crucial for the receptivity of the bovine uterus.


Assuntos
Endométrio , Células Epiteliais , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina , Cinurenina , Receptores de Hidrocarboneto Arílico , Triptofano Oxigenase , Triptofano , Animais , Bovinos , Feminino , Triptofano/farmacologia , Triptofano/metabolismo , Endométrio/metabolismo , Endométrio/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Cinurenina/metabolismo , Cinurenina/farmacologia , Triptofano Oxigenase/metabolismo , Triptofano Oxigenase/genética , Dinoprostona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/genética
19.
Front Immunol ; 15: 1425212, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39229264

RESUMO

Single-cell RNA sequencing (scRNA-seq) technology has emerged as a powerful tool for dissecting cellular heterogeneity and understanding the intricate biology of diseases, including cancer. Endometrial cancer (EC) stands out as the most prevalent gynecological malignancy in Europe and the second most diagnosed worldwide, yet its cellular complexity remains poorly understood. In this review, we explore the contributions of scRNA-seq studies to shed light on the tumor cells and cellular landscape of EC. We discuss the diverse tumoral and microenvironmental populations identified through scRNA-seq, highlighting the implications for understanding disease progression. Furthermore, we address potential limitations inherent in scRNA-seq studies, such as technical biases and sample size constraints, emphasizing the need for larger-scale research encompassing a broader spectrum of EC histological subtypes. Notably, a significant proportion of scRNA-seq analyses have focused on primary endometrioid carcinoma tumors, underscoring the need to incorporate additional histological and aggressive types to comprehensively capture the heterogeneity of EC. By critically evaluating the current state of scRNA-seq research in EC, this review underscores the importance of advancing towards more comprehensive studies to accelerate our understanding of this complex disease.


Assuntos
Neoplasias do Endométrio , Análise de Célula Única , Microambiente Tumoral , Humanos , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Análise de Célula Única/métodos , Microambiente Tumoral/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Análise de Sequência de RNA , Animais , Biomarcadores Tumorais/genética
20.
Ren Fail ; 46(2): 2369342, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39230047

RESUMO

Sepsis represents an organ dysfunction resulting from the host's maladjusted response to infection, and can give rise to acute kidney injury (AKI), which significantly increase the morbidity and mortality of septic patients. This study strived for identifying a novel therapeutic strategy for patients with sepsis-induced AKI (SI-AKI). Rat tubular epithelial NRK-52E cells were subjected to lipopolysaccharide (LPS) exposure for induction of in-vitro SI-AKI. The expressions of E1A binding protein p300 (EP300) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in NRK-52E cells were assessed by western blot and qRT-PCR, and their interaction was explored by chromatin immunoprecipitation performed with antibody for H3K27 acetylation (H3K27ac). The effect of them on SI-AKI-associated mitochondrial dysfunction of tubular epithelial cells was investigated using transfection, MTT assay, TUNEL staining, 2',7'-Dichlorodihydrofluorescein diacetate probe assay, Mitosox assay, and JC-1 staining. MTHFD2 and EP300 were upregulated by LPS exposure in NRK-52E cells. LPS increased the acetylation of H3 histone in the MTHFD2 promoter region, and EP300 suppressed the effect of LPS. EP300 ablation inhibited the expression of MTHFD2. MTHFD2 overexpression antagonized LPS-induced viability reduction, apoptosis promotion, reactive oxygen species overproduction, and mitochondrial membrane potential collapse of NRK-52E cells. By contrast, MTHFD2 knockdown and EP300 ablation brought about opposite consequences. Furthermore, MTHFD2 overexpress and EP300 ablation counteracted each other's effect in LPS-exposed NRK-52E cells. EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction of tubular epithelial cells in SI-AKI.


Assuntos
Injúria Renal Aguda , Proteína p300 Associada a E1A , Células Epiteliais , Lipopolissacarídeos , Metilenotetra-Hidrofolato Desidrogenase (NADP) , Mitocôndrias , Animais , Ratos , Acetilação , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Proteína p300 Associada a E1A/metabolismo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Células Epiteliais/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular , Histonas/metabolismo , Apoptose , Sepse/metabolismo , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Regulação para Cima
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