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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.
Nutrients ; 16(17)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39275261

RESUMO

Diabetic retinopathy (DR) is a specific microvascular problem of diabetes, which is mainly caused by hyperglycemia and may lead to rapid vision loss. Dietary polyphenols have been reported to decrease the risk of DR. Apocynum venetum L. leaves are rich in polyphenolic compounds and are popular worldwide for their health benefits as a national tea drink. Building on previous findings of antioxidant activity and aldose reductase inhibition of A. venetum, this study investigated the chemical composition of polyphenol-rich extract of A. venetum leaves (AVL) and its protective mechanism on ARPE-19 cells in hyperglycemia. Ninety-three compounds were identified from AVL by LC-MS/MS, including sixty-eight flavonoids, twenty-one organic acids, and four coumarins. AVL regulated the polyol pathway by decreasing the expression of aldose reductase and the content of sorbitol, enhancing the Na+K+-ATPase activity, and weakening intracellular oxidative stress effectively; it also could regulate the expression of autophagy-related proteins via the AMPK/mTOR/ULK1 signaling pathway to maintain intracellular homeostasis. AVL could restore the polyol pathway, inhibit oxidative stress, and maintain intracellular autophagy to protect cellular morphology and improve DR. The study reveals the phytochemical composition and protective mechanisms of AVL against DR, which could be developed as a functional food and/or candidate pharmaceutical, aiming for retina protection in diabetic retinopathy.


Assuntos
Apocynum , Autofagia , Glucose , Estresse Oxidativo , Extratos Vegetais , Folhas de Planta , Polifenóis , Epitélio Pigmentado da Retina , Humanos , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Polifenóis/análise , Folhas de Planta/química , Autofagia/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Glucose/metabolismo , Glucose/efeitos adversos , Apocynum/química , Estresse Oxidativo/efeitos dos fármacos , Polímeros , Linhagem Celular , Retinopatia Diabética/prevenção & controle , Retinopatia Diabética/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antioxidantes/farmacologia , Aldeído Redutase/metabolismo
3.
Nan Fang Yi Ke Da Xue Xue Bao ; 44(8): 1459-1466, 2024 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-39276041

RESUMO

OBJECTIVE: To investigate the mechanism by which conbercept reverses transforming growth factor-ß2 (TGF-ß2)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs). METHODS: Cultured HLEC SRA01/04 cells were treated with TGF-ß2, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α-SMA, and Snail), extracellular matrix components, and genes related to the TGF-ß/Smad signaling pathway. RESULTS: Conbercept significantly reduced TGF-ß2-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P <0.05). Transwell assay showed significantly lower cell migration ability in TGF-ß2+conbercept group than in TGF-ß2 group (P <0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-ß2-induced EMT (P <0.01). CONCLUSION: Conbercept inhibits TGF-ß2 induced EMT by downregulating the expression of pSmad2/3 in TGF-ß/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.


Assuntos
Proliferação de Células , Células Epiteliais , Transição Epitelial-Mesenquimal , Cristalino , Transdução de Sinais , Proteínas Smad , Fator de Crescimento Transformador beta2 , Humanos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Fator de Crescimento Transformador beta2/metabolismo , Cristalino/citologia , Cristalino/metabolismo , Proteínas Smad/metabolismo , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Caderinas/metabolismo , Apoptose/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular , Proteína Smad2/metabolismo
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 44(8): 1545-1552, 2024 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-39276050

RESUMO

OBJECTIVE: To investigate the mechanism by which swertiamarin (STM) ameliorates CD-like colitis in mice. METHODS: A Caco-2 cell model of TNF-α-stimulated apoptosis was established and divided into three groups: Con, TNF-α and STM, and the effects of STM on apoptosis and barrier function were assessed by Tunel staining, western blotting, immunofluorescence, and transepithelial electric resistance (TEER). A mouse model of 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) -induced CD-like colitis was established to assess the effects of STM on colitis, intestinal barrier function and epithelial cell apoptosis. The regulatory role of the PI3K/AKT pathway in STM-induced resistance to intestinal epithelial cell apoptosis was investigated in both the cell model and mouse models. RESULTS: TUNEL staining showed that in Caco-2 cells with TNF-α stimulation, STM treatment significantly reduced the percentage of TUNEL-stained cells (P<0.05). STM obviously reduced TNF-α-induced enhancement of cleaved-caspase 3 and Bax expressions (P<0.05), increased Bcl-2 expression (P<0.05), protected intestinal barrier integrity and function by restoring transepithelial electrical resistance (TEER) of the cells, promoted normal localization and expressions of the tight junction proteins (ZO1 and claudin 1) (P<0.05), and inhibited the expression of pro-inflammatory factors (IL-6 and CCL3) (P<0.05) in TNF-α-stimulated Caco-2 cells. In the mouse models, STM significantly alleviated TNBS-induced CD-like colitis and intestinal barrier dysfunction (P<0.05) as shown by improved weight loss, lowered Disease Activity Index (DAI) score and inflammation score, reduction of IL-6 and CCL3 release, and restoration of intestinal barrier permeability, colonic TEER, bacterial translocation, and localization and expressions of the tight junction proteins. Mechanistically, STM inhibited the expressions of p-PI3K and p-AKT in both the cell model and mouse model(P<0.05), and treatment with 740Y-P (a PI3K/AKT pathway activator) significantly attenuated the inhibitory effect of STM on TNF-α-induced apoptosis in Caco-2 cells (P<0.05). CONCLUSION: STM inhibits intestinal epithelial cell apoptosis at least in part by suppressing activation of the PI3K/AKT pathway to ameliorate intestinal barrier dysfunction and colitis in mice.


Assuntos
Apoptose , Colite , Células Epiteliais , Ácido Trinitrobenzenossulfônico , Fator de Necrose Tumoral alfa , Animais , Camundongos , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Apoptose/efeitos dos fármacos , Humanos , Células CACO-2 , Fator de Necrose Tumoral alfa/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glucosídeos Iridoides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Caspase 3/metabolismo
5.
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
6.
Int J Mol Sci ; 25(17)2024 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-39273173

RESUMO

Escherichia coli O157:H7 (E. coli O157) is known for causing severe foodborne illnesses such as hemorrhagic colitis and hemolytic uremic syndrome. Although E. coli O157 is typically regarded as an extracellular pathogen and a weak biofilm producer, some E. coli O157 strains, including a clinical strain ATCC 43895, exhibit a notable ability to invade bovine crypt cells and other epithelial cells, as well as to form robust biofilm. This invasive strain persists in the bovine host significantly longer than non-invasive strains. Various surface-associated factors, including lipopolysaccharides (LPS), flagella, and other adhesins, likely contribute to this enhanced invasiveness and biofilm formation. In this study, we constructed a series of LPS-core deletion mutations (waaI, waaG, waaF, and waaC) in E. coli O157 ATCC 43895, resulting in stepwise truncations of the LPS. This approach enabled us to investigate the effects on the biosynthesis of key surface factors, such as flagella and curli, and the ability of this invasive strain to invade host cells. We confirmed the LPS structure and found that all LPS-core mutants failed to form biofilms, highlighting the crucial role of core oligosaccharides in biofilm formation. Additionally, the LPS inner-core mutants ΔwaaF and ΔwaaC lost the ability to produce flagella and curli. Furthermore, these inner-core mutants exhibited a dramatic reduction in adherence to and invasion of epithelial cells (MAC-T), showing an approximately 100-fold decrease in cell invasion compared with the outer-core mutants (waaI and waaG) and the wild type. These findings underscore the critical role of LPS-core truncation in impairing flagella and curli biosynthesis, thereby reducing the invasion capability of E. coli O157 ATCC 43895.


Assuntos
Biofilmes , Escherichia coli O157 , Flagelos , Lipopolissacarídeos , Flagelos/metabolismo , Flagelos/genética , Lipopolissacarídeos/biossíntese , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Escherichia coli O157/fisiologia , Biofilmes/crescimento & desenvolvimento , Animais , Bovinos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Aderência Bacteriana , Células Epiteliais/microbiologia , Células Epiteliais/metabolismo
7.
Int J Mol Sci ; 25(17)2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39273216

RESUMO

Galectins have the potential to interact with transmembrane glycoproteins to modulate their functions. Since galectin-1 interacts with PDGF-Rß, we analyzed the effect of galectin-1 on PDGF-BB-mediated AKT signaling in primary human retinal pigment epithelial (RPE) cells and galectin-1-deficient immortalized human RPE cells (LGALS1-/-/ARPE-19) following incubation with PDGF-BB and galectin-1. Expression and localization of galectin-1, PDGF-Rß and pAKT were investigated using western blot analysis and immunohistochemical staining. Cell proliferation of RPE cells was analyzed using BrdU ELISA. Following treatment of human RPE cells with human recombinant (hr)-galectin-1 and PDGF-BB, an intense clustering of PDGF-Rß and colocalization with galectin-1 were detected. By Western blot analysis and immunocytochemistry of human RPE cells, an enhanced PDGF-BB-mediated expression of pAKT was observed, which was substantially reduced by additional incubation with hr-galectin-1. Vice versa, in LGALS1-/-/ARPE-19 cells, the PDGF-BB-induced pAKT signal was enhanced compared to wild-type cells. Furthermore, a decreased expression of PDGF-Rß in human RPE cells was observed after treatment with PDGF-BB and hr-galectin-1, while in untreated LGALS1-/-/ARPE-19 cells, its constitutive expression was increased. In addition, after treatment of RPE cells with hr-galectin-1, the PDGF-BB-induced proliferation was markedly reduced. In summary, galectin-1 has the distinct potential to reduce PDGF-mediated pAKT signaling and proliferation in human RPE cells-an effect that is most likely facilitated via a decreased expression of PDGF-Rß.


Assuntos
Becaplermina , Proliferação de Células , Galectina 1 , Proteínas Proto-Oncogênicas c-akt , Epitélio Pigmentado da Retina , Transdução de Sinais , Humanos , Galectina 1/metabolismo , Galectina 1/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Becaplermina/metabolismo , Becaplermina/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Linhagem Celular , Células Epiteliais/metabolismo
8.
Int J Mol Sci ; 25(17)2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39273233

RESUMO

To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6. IPA analysis of these DEGs suggested that FABP5 may be involved in essential roles required for cell development, cell survival and cell homeostasis. In support of this possibility, both mitochondrial and glycolytic functions of HNPCE cells, in which mRNA expression of FABP5, but not that of FABP7, was detected, were shown by using a Seahorse XFe96 Bioanalyzer to be dramatically suppressed by FABP ligand 6-induced inhibition of the activity of FABP5. Furthermore, in IPA upstream analysis, various unfolded protein response (UPR)-related factors were identified as upstream and causal network master regulators. Analysis by qPCR analysis showed significant upregulation of the mRNA expression of most of UPR-related factors and aquaporin1 (AQP1). The findings in this study suggest that HNPCE is one of intraocular cells producing FABP5 and may be involved in the maintenance of UPR and AQP1-related functions of HNPCE.


Assuntos
Proteínas de Ligação a Ácido Graxo , Humanos , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Linhagem Celular , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/citologia , Células Epiteliais/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Regulação da Expressão Gênica , Corpo Ciliar/metabolismo , Corpo Ciliar/citologia , Glicólise
9.
Int J Mol Sci ; 25(17)2024 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-39273267

RESUMO

Newborn lambs are susceptible to pathogenic bacterial infections leading to enteritis, which affects their growth and development and causes losses in sheep production. It has been reported that N6-methyladenosine (m6A) is closely related to innate immunity, but the effect of m6A on sheep small intestinal epithelial cells (IECs) and the mechanism involved have not been elucidated. Here, we investigated the effects of m6A on lipopolysaccharide (LPS)-induced inflammatory responses, apoptosis and oxidative stress in primary sheep IECs. First, the extracted IECs were identified by immunofluorescence using the epithelial cell signature protein cytokeratin 18 (CK18), and the cellular activity of IECs induced by different concentrations of LPS was determined by the CCK8 assay. Meanwhile, LPS could induce the upregulation of mRNA and protein levels of IECs cytokines IL1ß, IL6 and TNFα and the apoptosis marker genes caspase-3, caspase-9, Bax, and apoptosis rate, reactive oxygen species (ROS) levels and mRNA levels of CAT, Mn-SOD and CuZn-SOD, and METTL3 were found to be upregulated during induction. It was hypothesized that METTL3 may have a potential effect on the induction of IECs by LPS. Overexpression and knockdown of METTL3 in IECs revealed that a low-level expression of METTL3 could reduce the inflammatory response, apoptosis and ROS levels in LPS-induced IECs to some extent. The results suggest that METTL3 may be a genetic marker for potential resistance to cellular damage.


Assuntos
Apoptose , Células Epiteliais , Intestino Delgado , Lipopolissacarídeos , Metiltransferases , Animais , Lipopolissacarídeos/toxicidade , Ovinos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Metiltransferases/metabolismo , Metiltransferases/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Intestino Delgado/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Citocinas/metabolismo , Citocinas/genética , Células Cultivadas
11.
Sci Rep ; 14(1): 21050, 2024 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-39251689

RESUMO

Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female.


Assuntos
Infecções por Chlamydia , Chlamydia trachomatis , Citocinas , Imunidade nas Mucosas , Neutrófilos , Sêmen , Chlamydia trachomatis/imunologia , Chlamydia trachomatis/fisiologia , Humanos , Feminino , Sêmen/imunologia , Sêmen/microbiologia , Sêmen/metabolismo , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Citocinas/metabolismo , Masculino , Células Epiteliais/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Fagocitose , Colo do Útero/microbiologia , Colo do Útero/imunologia
12.
BMC Nephrol ; 25(1): 297, 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39251943

RESUMO

BACKGROUND: Diabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it's role in HG-induced TECs remains to be elucidated. METHODS: The HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFß/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB. RESULTS: HG induced an increase in the expression of P4HB and TGFß, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFß/SMAD3-related proteins in HK-2 cells. CONCLUSION: P4HB regulated the TGFß/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells.


Assuntos
Células Epiteliais , Transição Epitelial-Mesenquimal , Fibrose , Glucose , Túbulos Renais , Proteína-Arginina N-Metiltransferases , Proteínas Repressoras , Transdução de Sinais , Proteína Smad3 , Fator de Crescimento Transformador beta , Humanos , Proteína Smad3/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glucose/farmacologia , Glucose/toxicidade , Glucose/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Fator de Crescimento Transformador beta/metabolismo , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Linhagem Celular , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Espécies Reativas de Oxigênio/metabolismo
13.
Cell Genom ; 4(9): 100652, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39265526

RESUMO

Missing regulatory effects of asthma genetic risks might be hidden within specific cell states. In this issue of Cell Genomics, Djeddi et al.1 uncover how airway epithelial cells, when activated by rhinovirus, influence genetic susceptibility to childhood-onset asthma, and this preview emphasizes the need to address these missing regulatory effects across diverse cell states.


Assuntos
Asma , Rhinovirus , Humanos , Asma/virologia , Asma/genética , Asma/epidemiologia , Asma/etiologia , Rhinovirus/genética , Criança , Predisposição Genética para Doença , Células Epiteliais/virologia , Células Epiteliais/patologia , Idade de Início
14.
Physiol Rep ; 12(17): e70038, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39238069

RESUMO

Osteopontin (OPN) is a multi-functional glycoprotein that coordinates the innate immune response, prevents nanocrystal formation in renal tubule fluid, and is a biomarker for kidney injury. OPN expression is markedly increased in cystic epithelial cells of polycystic kidney disease (PKD) kidneys; however, its role in PKD progression remains unclear. We investigated the in vitro effects of recombinant OPN on the proliferation of tubular epithelial cells from PKD and normal human kidneys and in vivo effects of OPN deletion on kidney cyst formation, fibrosis, and mineral metabolism in pcy/pcy mice, a non-orthologous model of autosomal-dominant PKD. In vitro studies revealed that OPN enhanced the proliferation of PKD cells but had no effect on normal kidney cells. Deletion of OPN in pcy/pcy mice significantly reduced kidney cyst burden; however, this was accompanied by increased fibrosis and no change in kidney function. The loss of OPN had no effect on kidney macrophage numbers, cyst epithelial cell proliferation, or apoptosis. Furthermore, there was no difference in kidney mineral deposition or mineral metabolism parameters between pcy/pcy mice with and without OPN expression. Global deletion of OPN reduced kidney cyst burden, while paradoxically exacerbating kidney fibrosis in mice with cystic kidney disease.


Assuntos
Fibrose , Osteopontina , Animais , Feminino , Humanos , Masculino , Camundongos , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Rim/metabolismo , Rim/patologia , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteopontina/metabolismo , Osteopontina/genética , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Doenças Renais Policísticas/genética
15.
Stem Cell Res Ther ; 15(1): 293, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39256850

RESUMO

BACKGROUND: Understanding the role of cytokines in tooth development is critical for advancing dental tissue engineering. Fibroblast growth factor 9 (FGF9) is the only FGF consistently expressed throughout dental epithelial tissue, from the initiation of tooth bud formation to tooth maturation. However, mice lacking Fgf9 (Fgf9-/-) surprisingly show no obvious abnormalities in tooth development, suggesting potential compensation by other FGFs. Here we report findings from an Fgf9S99N mutation mouse model, a loss-of-function mutation with a dominant negative effect. Our study reveals that Fgf9 is crucial for dental epithelial stem cell (DESC) survival and enamel formation. METHODS: To dissect the role of Fgf9 in tooth development, we performed the micro-CT, histomorphological analysis and gene expression assay in mice and embryos with S99N mutation. In addition, we assessed the effect of FGF9 on the DESC survival and dental epithelial differentiation by DESC sphere formation assay and tooth explant culture. Cell/tissue culture methods, gene expression analysis, specific inhibitors, and antibody blockage analysis were employed to explore how Fgf9 regulates enamel differentiation and DESC survival through both direct and indirect mechanisms. RESULTS: The Fgf9S99N mutation in mice led to reduced ameloblasts, impaired enamel formation, and increased apoptosis in the cervical loop (CL). DESC sphere culture experiments revealed that FGF9 facilitated DESC survival via activating ERK/CREB signaling, without affecting cell proliferation. Furthermore, in vitro tissue culture experiments demonstrated that FGF9 promoted enamel formation in a manner dependent on the presence of mesenchyme. Interestingly, FGF9 stimulation inhibited enamel formation in isolated enamel epithelia and DESC spheres. Further investigation revealed that FGF9 supports DESC survival and promotes amelogenesis by stimulating the secretion of FGF3 and FGF10 in dental mesenchymal cells via the MAPK/ERK signaling pathway. CONCLUSIONS: Our study demonstrates that Fgf9 is essential for DESC survival and enamel formation. Fgf9 performs as a dual-directional regulator of the dental enamel epithelium, not only inhibiting DESC differentiation into ameloblasts to preserve the stemness of DESC, but also promoting ameloblast differentiation through epithelial-mesenchymal interactions.


Assuntos
Esmalte Dentário , Células Epiteliais , Fator 9 de Crescimento de Fibroblastos , Células-Tronco , Animais , Fator 9 de Crescimento de Fibroblastos/metabolismo , Fator 9 de Crescimento de Fibroblastos/genética , Camundongos , Esmalte Dentário/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Células Epiteliais/metabolismo , Incisivo/metabolismo , Sobrevivência Celular , Diferenciação Celular
16.
Brain Behav ; 14(9): e3648, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39262161

RESUMO

BACKGROUND: The blood-cerebrospinal fluid barrier (BCSFB) comprises the choroid plexus epithelia. It is important for brain development, maintenance, function, and especially for maintaining immune homeostasis in the cerebrospinal fluid (CSF). Although previous studies have shown that the peripheral immune function of the body is impaired upon exposure to microgravity, no studies have reported changes in immune cells and cytokines in the CSF that reflect neuroimmune status. The purpose of this study is to investigate the alterations in cerebrospinal fluid (CSF) immune homeostasis induced by microgravity and its mechanisms. This research is expected to provide basic data for brain protection of astronauts during spaceflight. METHODS: The proportions of immune cells in the CSF and peripheral blood (PB) of SMG rats were analyzed using flow cytometry. Immune function was evaluated by measuring cytokine concentrations using the Luminex method. The histomorphology and ultrastructure of the choroid plexus epithelia were determined. The concentrations of intercellular junction proteins in choroid plexus epithelial cells, including vascular endothelial-cadherin (VE-cadherin), zonula occludens 1 (ZO-1), Claudin-1 and occludin, were detected using western blotting and immunofluorescence staining to characterize BCSFB injury. RESULTS: We found that SMG caused significant changes in the proportion of CD4 and CD8 T cells in the CSF and a significant increase in the levels of cytokines (GRO/KC, IL-18, MCP-1, and RANTES). In the PB, there was a significant decrease in the proportion of T cells and NKT cells and a significant increase in cytokine levels (GRO/KC, IL-18, MCP-1, and TNF-α). Additionally, we observed that the trends in immune markers in the PB and CSF were synchronized within specific SMG durations, suggesting that longer SMG periods (≥21 days) have a more pronounced impact on immune markers. Furthermore, 21d-SMG resulted in ultrastructural disruption and downregulated expression of intercellular junction proteins in rat choroid plexus epithelial cells. CONCLUSIONS: We found that SMG disrupts the BCSFB and affects the CSF immune homeostasis. This study provides new insights into the health protection of astronauts during spaceflight.


Assuntos
Barreira Hematoencefálica , Plexo Corióideo , Citocinas , Homeostase , Simulação de Ausência de Peso , Animais , Homeostase/fisiologia , Ratos , Plexo Corióideo/imunologia , Plexo Corióideo/metabolismo , Masculino , Citocinas/metabolismo , Citocinas/líquido cefalorraquidiano , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/imunologia , Líquido Cefalorraquidiano/imunologia , Líquido Cefalorraquidiano/metabolismo , Ratos Sprague-Dawley , Células Epiteliais/metabolismo , Células Epiteliais/imunologia
17.
Immunity ; 57(9): 2002-2004, 2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39260351

RESUMO

Intestinal macrophages play a key role in regulating immune tolerance in the gut. In this issue of Immunity, Mertens et al. uncover a mechanism for the establishment of memory in macrophage tolerance in the gut involving a bistable metabolic switch in macrophages and an intercellular positive feedback between macrophages and intestinal epithelial cells (IECs).


Assuntos
Tolerância Imunológica , Mucosa Intestinal , Macrófagos , Macrófagos/imunologia , Tolerância Imunológica/imunologia , Humanos , Animais , Mucosa Intestinal/imunologia , Retroalimentação Fisiológica , Intestinos/imunologia , Células Epiteliais/imunologia
18.
PeerJ ; 12: e17998, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39253600

RESUMO

Age related cataracts (ARC) represent the main reason for blindness globally. The lens epithelial cells (LECs) participate not only in the metabolism of many substances in the lens but also in maintaining lens transparency. This study used lipidomics to investigate the metabolic differences in LECs of ARC patients with different severity, aiming at identifying potential metabolic biomarkers of ARC. Patients diagnosed with ARC and underwent cataract surgery at Shanghai Tongren Hospital were selected to participate in this study, which were classified as mild ARC group and severe ARC group. During their cataract surgery, anterior lens capsules(LCs) containing LECs were obtained. The lipidomics of LECs were analyzed using the liquid chromatography­mass spectrometry (LC-MS). Potential pathways of lipids were searched for using databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst platform. In LEC lipids, 26 lipids have been identified as potential biomarkers between mild ARC and severe ARC, with AUC values of 0.67-0.94. The pathway analysis results revealed that the Glycerophospholipid (GPL) metabolism was significantly influenced, indicating that these metabolic markers contribute significantly to regulating this pathway. The LEC metabolic spectrum demonstrates a proficient ability to differentiate between patients with varying levels of cataracts. Herein, we have successfully identified potential metabolic biomarkers and pathways that have proven to be valuable in enhancing our understanding of ARC pathogenesis. The finding has translational value for developing new cataract treatment methods in the future.


Assuntos
Catarata , Células Epiteliais , Cristalino , Lipidômica , Humanos , Catarata/metabolismo , Catarata/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Masculino , Feminino , Idoso , Cristalino/metabolismo , Cristalino/patologia , Biomarcadores/metabolismo , Biomarcadores/análise , Pessoa de Meia-Idade , Metabolismo dos Lipídeos , Cromatografia Líquida
19.
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
20.
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
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