RESUMO
Self-assembled DNA nanostructures hold great promise in biosensing, drug delivery and nanomedicine. Nevertheless, challenges like instability and inefficiency in cellular uptake of DNA nanostructures under physiological conditions limit their practical use. To tackle these obstacles, this study proposes a novel approach that integrates the cationic polymer polyethyleneimine (PEI) with DNA self-assembly. The hypothesis is that the positively charged linear PEI can facilitate the self-assembly of DNA nanostructures, safeguard them against harsh conditions and impart them with the cellular penetration characteristic of PEI. As a demonstration, a DNA nanotube (PNT) was successfully synthesized through PEI mediation, and it exhibited significantly enhanced stability and cellular uptake efficiency compared to conventional Mg2+-assembled DNA nanotubes. The internalization mechanism was further found to be both clathrin-mediated and caveolin-mediated endocytosis, influenced by both PEI and DNA. To showcase the applicability of this hybrid nanostructure for biomedical settings, the KRAS siRNA-loaded PNT was efficiently delivered into lung adenocarcinoma cells, leading to excellent anticancer effects in vitro. These findings suggest that the PEI-mediated DNA assembly could become a valuable tool for future biomedical applications.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Nanotubos , Polietilenoimina , Proteínas Proto-Oncogênicas p21(ras) , RNA Interferente Pequeno , Humanos , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , DNA/química , Portadores de Fármacos/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Nanotubos/química , Tamanho da Partícula , Polietilenoimina/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologiaRESUMO
Garlic has a long history to be used for medicine and food purposes. Black garlic, the fermented product of fresh garlic, is considered with better biological activities, such as antioxidant activity, and is developed as an increasingly popular functional food. Polysaccharides are the major components of fresh and black garlic, and immunomodulatory activity is one major pharmacological effect of polysaccharides. Therefore, chemical characteristics and immunomodulatory effects of polysaccharides from fresh and black garlic are investigated and compared in vitro for the 1st time, in order to reveal their molecular and pharmacological differences. It is demonstrated that the molecular weights of polysaccharides from the 2 sources and molar ratios of monosaccharides after acid hydrolysis are greatly variant. The effects of polysaccharides from 2 sources on RAW 264.7 macrophages functions, including promotion of phagocytosis, release of NO, and expressions of several immune-related cytokines (including interleukin [IL]-6, IL-10, tumor necrosis factor alpha, and interferon gamma), were different from each other. The results indicated that fresh garlic polysaccharide exhibited stronger immunomodulatory activities than that of black garlic. Moreover, it is revealed that fructan might be the bioactive component in garlic and it is indicated that during the fermentation treatment, fructan constituents of garlic has degraded, and basically no immunomodulatory effect can be found in black garlic polysaccharides.
Assuntos
Citocinas/metabolismo , Fermentação , Alho/química , Fatores Imunológicos/farmacologia , Macrófagos/efeitos dos fármacos , Polissacarídeos/farmacologia , Animais , Frutanos/farmacologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Peso Molecular , Monossacarídeos/análise , Óxido Nítrico/metabolismo , Fagocitose/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Raízes de Plantas , Polissacarídeos/química , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) is a counter-regulator against ACE by converting angiotensin II (Ang-II) to Ang-(1-7), but the effect of ACE2 and Ang-(1-7) on endothelial cell function and atherosclerotic evolution is unknown. We hypothesized that ACE2 overexpression and Ang-(1-7) may protect endothelial cell function by counterregulation of angiotensin II signaling and inhibition of inflammatory response. METHODS: We used a recombinant adenovirus vector to locally overexpress ACE2 gene (Ad-ACE2) in human endothelial cells in vitro and in apoE-deficient mice in vivo. The Ang II-induced MCP-1, VCAM-1 and E-selectin expression, endothelial cell migration and adhesion of human monocytic cells (U-937) to HUVECs by ACE2 gene transfer were evaluated in vitro. Accelerated atherosclerosis was studied in vivo, and atherosclerosis was induced in apoE-deficient mice which were divided randomly into four groups that received respectively a ACE2 gene transfer, Ad-ACE2, Ad-EGFP, Ad-ACE2 + A779, an Ang-(1-7) receptor antagonist, control group. After a gene transfer for 4 weeks, atherosclerotic pathology was evaluated. RESULTS: ACE2 gene transfer not only promoted HUVECs migration, inhibited adhesion of monocyte to HUVECs and decreased Ang II-induced MCP-1, VCAM-1 and E-selectin protein production in vitro, but also decreased the level of MCP-1, VCAM-1 and interleukin 6 and inhibit atherosclerotic plaque evolution in vivo. Further, administration of A779 increased the level of MCP-1, VCAM-1 and interleukin 6 in vivo and led to further advancements in atherosclerotic extent. CONCLUSIONS: ACE2 and Ang-(1-7) significantly inhibit early atherosclerotic lesion formation via protection of endothelial function and inhibition of inflammatory response.
Assuntos
Angiotensina II/fisiologia , Angiotensina I/fisiologia , Aterosclerose/prevenção & controle , Endotélio Vascular/fisiologia , Inflamação/prevenção & controle , Fragmentos de Peptídeos/fisiologia , Peptidil Dipeptidase A/fisiologia , Transdução de Sinais/fisiologia , Angiotensina I/genética , Enzima de Conversão de Angiotensina 2 , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/fisiopatologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Quimiocina CCL2/fisiologia , Modelos Animais de Doenças , Selectina E/fisiologia , Endotélio Vascular/citologia , Técnicas de Transferência de Genes , Humanos , Técnicas In Vitro , Inflamação/fisiopatologia , Camundongos , Fragmentos de Peptídeos/genética , Peptidil Dipeptidase A/genética , Molécula 1 de Adesão de Célula Vascular/fisiologiaRESUMO
OBJECTIVES: The aim of this study was to test the hypothesis that angiotensin (Ang)-converting enzyme-2 (ACE2) overexpression may inhibit myocardial collagen accumulation and improve left ventricular (LV) remodeling and function in diabetic cardiomyopathy. BACKGROUND: Hyperglycemia activates the renin-Ang system, which promotes the accumulation of extracellular matrix and progression of cardiac remodeling and dysfunction. METHODS: Ninety male Wistar rats were divided randomly into treatment (n = 80) and control (n = 10) groups. Diabetes was induced in the treatment group by a single intraperitoneal injection of streptozotocin. Twelve weeks after streptozotocin injection, rats in the treatment group were further divided into adenovirus-ACE2, adenovirus-enhanced green fluorescent protein, losartan, and mock groups (n = 20 each). LV volume; LV systolic and diastolic function; extent of myocardial fibrosis; protein expression levels of ACE2, Ang-converting enzyme, and Ang-(1-7); and matrix metalloproteinase-2 activity were evaluated. Cardiac myocyte and fibroblast culture was performed to assess Ang-II and collagen protein expression before and after ACE2 gene transfection. RESULTS: Four weeks after ACE2 gene transfer, the adenovirus-ACE2 group showed increased ACE2 expression, matrix metalloproteinase-2 activity, and LV ejection fractions and decreased LV volumes, myocardial fibrosis, and ACE, Ang-II, and collagen expression in comparison with the adenovirus-enhanced green fluorescent protein and control groups. ACE2 was superior to losartan in improving LV remodeling and function and reducing collagen expression. The putative mechanisms may involve a shift in balance toward an inhibited fibroblast-myocyte cross-talk for collagen and transforming growth factor-beta production and enhanced collagen degradation by matrix metalloproteinase-2. CONCLUSIONS: ACE2 inhibits myocardial collagen accumulation and improves LV remodeling and function in a rat model of diabetic cardiomyopathy. Thus, ACE2 provides a promising approach to the treatment of patients with diabetic cardiomyopathy.
Assuntos
Cardiomiopatias Diabéticas/genética , Regulação da Expressão Gênica , Miocárdio/enzimologia , Peptidil Dipeptidase A/genética , RNA Mensageiro/genética , Função Ventricular Esquerda/fisiologia , Remodelação Ventricular/fisiologia , Enzima de Conversão de Angiotensina 2 , Animais , Western Blotting , Diabetes Mellitus Experimental , Cardiomiopatias Diabéticas/enzimologia , Cardiomiopatias Diabéticas/fisiopatologia , Ecocardiografia , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/fisiopatologia , Imuno-Histoquímica , Masculino , Miocárdio/patologia , Peptidil Dipeptidase A/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: Angiotensin converting enzyme 2 (ACE2) efficiently hydrolyses the potent vasoconstrictor angiotensin II to vasodilative angiotensin (1-7). We hypothesized that ACE2 overexpression may inhibit inflammation response in atherosclerotic plaque by degrading Ang II into Ang-(1-7). METHODS: Atherosclerosis (AS) plaques were induced in the abdominal aorta of 38 rabbits by endothelial injury and atherogenic diet for 3 months. Rabbits were then underwent injection of a recombinant adenovirus (2.5 x 10(9) pfu/ml) carrying a murine ACE2 gene (Ad-ACE2) through a catheter into the abdominal aortic segments rich in plaques (n = 19) or injection of a control vector Ad-EGFP (n = 19). One month later, all rabbits were sacrificed and plaques from aortic segments were analyzed. RESULTS: ACE2 expression in aortic tissues of the Ad-ACE2 group were confirmed by immunohistochemistry. Macrophage infiltration (13.6% +/- 4.2% vs. 23.6% +/- 6.9%, P < 0.01) and MCP-1 expression (13.2% +/- 0.4% vs. 25.0% +/- 7.4%, P < 0.01) were significantly reduced in Ad-ACE2 group compared to Ad-EGFP group. CONCLUSIONS: Overexpression of ACE2 inhibited atherosclerotic plaque inflammation response in hypercholesterolemic rabbits.
Assuntos
Aterosclerose/genética , Peptidil Dipeptidase A/genética , Transfecção , Enzima de Conversão de Angiotensina 2 , Animais , Aterosclerose/metabolismo , Células Cultivadas , Dieta Aterogênica , Vetores Genéticos , CoelhosRESUMO
OBJECTIVE: The purpose of this study was to test the hypothesis that ACE2 overexpression may enhance atherosclerotic plaque stability by antagonizing ACE activity and converting angiotensin II to angiotensin 1-7. METHODS AND RESULTS: Atherosclerotic plaques were induced in the abdominal aorta of 114 rabbits by endothelial injury and atherogenic diet. Gene therapy was performed in group A at week 4 and in group B at week 12, respectively. Each group of rabbits were randomly divided into 3 subgroups which received, respectively, a recombinant ACE2 expressing vector (AdACE2), a control vector AdEGFP and AdACE2+A779, an antagonist of angiotensin 1-7 receptor. Local ACE2 overexpression attenuated the progression of lesions from week 4 to week 8, but not progression of plaque size from week 12 to week 16. In group B rabbits, local ACE2 overexpression resulted in stable plaque compositions, ie, fewer macrophages, less lipid deposition and more collagen contents, higher plaque stability scores, decreased angiotensin II levels, and increased angiotensin 1-7 levels in plaque tissues in the AdACE2 subgroup compared with those in the AdEGFP subgroup. CONCLUSIONS: Overexpression of ACE2 results in stabilized atherosclerotic plaques and the mechanism is probably the conversion of vasoconstrictive angiotensin II to vessel protective angiotensin 1-7.