RESUMO
In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.
Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Survivina/genética , Biomarcadores , Testes ImediatosRESUMO
To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.
Assuntos
Aminoácidos , Corantes Fluorescentes , Nanopartículas , Placa Aterosclerótica , Espécies Reativas de Oxigênio , Placa Aterosclerótica/diagnóstico por imagem , Animais , Espécies Reativas de Oxigênio/metabolismo , Corantes Fluorescentes/química , Nanopartículas/química , Camundongos , Aminoácidos/química , Humanos , Medição de Risco , Imagem Óptica , Raios Infravermelhos , Células RAW 264.7RESUMO
Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.
Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , MicroRNAs , Sistemas CRISPR-Cas/genética , Técnicas Biossensoriais/métodos , Humanos , MicroRNAs/análise , MicroRNAs/metabolismo , Regulação Alostérica , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células HEK293RESUMO
The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 µM, and 8.3 × 10-5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.
Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Bioensaio , Processamento de Imagem Assistida por Computador , OligonucleotídeosRESUMO
The fat mass and obesity-associated enzyme (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) residues in mRNA, regulates the cellular level of m6A modification, and plays a critical role in human obesity and cancers. Herein, we develop a single-quantum-dot (QD)-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of m6A can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the QD surface, enabling the occurrence of FRET between the QD and Cy5. The presence of inhibitors can inhibit the FTO demethylation and consequently abolish FRET between the QD and Cy5. The inhibition effect of inhibitors upon FTO demethylation can be simply evaluated by monitoring the decrease of Cy5 counts. We use this nanosensor to screen several small-molecule inhibitors and identify diacerein as a highly selective inhibitor of FTO. Diacerein can inhibit the demethylation activity of endogenous FTO in HeLa cells. Interestingly, diacerein is neither a structural mimic of 2-oxoglutarate (2-OG) nor a chelator of metal ions, and it can selectively inhibit FTO demethylation by competitively binding the m6A-containing substrate.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Técnicas Biossensoriais/métodos , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência , Pontos Quânticos/química , Adenosina/análogos & derivados , Adenosina/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Sítios de Ligação , Carbocianinas/química , Domínio Catalítico , Desmetilação do DNA , Inibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Simulação de Dinâmica Molecular , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Ferulic acid (FA) is an active component of the traditional Chinese herb Angelica sinensis. Numerous health benefits have been attributed to FA, but few studies have investigated the effects of FA on osteoblasts (Obs). Our work studied the effects of FA on proliferation, differentiation, and mineralization of rat calvarial Obs and examined the signaling pathways involved. Cell proliferation and differentiation were evaluated by Cell Counting Kit-8 (CCK-8) and alkaline phosphatase (ALP) assay kit, respectively. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKGII) expression was silenced by small interfering RNA (siRNA). The mRNA expression was investigated by semi-quantitative PCR. FA (40-2560 µM) promoted Ob proliferation and differentiation; at 40-640 µM, FA stimulated calcified nodule formation and increased the expression of osteogenic genes encoding osteopontin and collagen-l. FA (40-2560 µM) increased cGMP levels in Obs and upregulated the expression of PKGII, EnaCα, and ENaCγ mRNAs. Downregulated ENaCα mRNA expression in Obs transfected with the siRNA for PKGII was reversed when FA was introduced into Obs. These results demonstrated that FA promoted proliferation, differentiation, and mineralization of Obs in vitro, and enhanced osteogenic genes expression partly through the cGMP-PKGII-ENaC signaling pathway.
Assuntos
Proliferação de Células/efeitos dos fármacos , Ácidos Cumáricos/farmacologia , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular , GMP Cíclico/metabolismo , Estrutura Molecular , Osteogênese/efeitos dos fármacos , Osteopontina , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
To determine the optimum aqueous extract protocol for Yugubao traditional Chinese medicines formula by using orthogonal experiment design. Through serum pharmacology research, L9(34) orthogonal design with single factor investigation was used to optimize the aqueous extract protocol for Yugubao formula. The effect of water extraction on activity of alkaline phosphatase (ALP) in osteoblast was referred as the evaluation index for investigating four factors: water consumption (A), heating time (B), soaking time (C), and number of decocting (D), analyzing the optimum extraction conditions, and verifying the effectiveness of this process. The optimum aqueous extract protocol for Yugubao was as follows: adding 8 times water into Chinese medical materials, heating for 60 min, soaking for 30 min, and decocting for 1 time. The drug serum of this aqueous extract of Yugubao could significantly up-regulate the osteogenic genes expression. The optimum aqueous extract protocol for Yugubao formula was established in this experiment, providing evidence for the development and utilization of Yugubao traditional Chinese medicines formula.
Assuntos
Fracionamento Químico/métodos , Medicamentos de Ervas Chinesas/química , Extratos Vegetais/química , Medicina Tradicional Chinesa , Projetos de Pesquisa , Tecnologia Farmacêutica , ÁguaRESUMO
OBJECTIVE: To study the influence of Plantaginis Semen on cell proliferation, differentiation and function of rat osteoblasts, and investigate the regulation effects of rat osteoblast epithelial sodium channel (ENaC) on bone formation. METHODS: The animal serum was prepared by serum pharmacology means. The cells were got by separating and inducing the SD neonatal rat's skull bone. Cell proliferation and differentiation were evaluated by CCK-8 assay kit and AKP assay kit respectively. Regulation effects on mRNA expression of ENaC and osteogenesis gene were investigated by semi-quantitative PCR. RESULTS: Plantaginis Semen stimulated the osteoblasts proliferation and differentiation,the difference between treatment group and control group had statistical significance (P < 0.01) in a dose-dependent manner. The effects of Plantaginis Semen serum on alpha-ENaC gene expression paralleled those on osteogenic gene (OC, ALP, OP) expression level. CONCLUSION: Plantaginis Semen stimulates proliferation, differentiation and the mRNA expression of ENaC and osteogenesis gene in rat osteoblasts. Our results suggest that ENaC participate in the effects of Plantaginis Semen serum on osteoblast bone formation. Regulation of ENaC channel expression and function may provide a new clue for research on treatment of osteoporosis with traditional Chinese medicine.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Canais Epiteliais de Sódio/metabolismo , Osteoblastos/efeitos dos fármacos , Osteogênese , Plantago/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/isolamento & purificação , Canais Epiteliais de Sódio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/metabolismo , Ratos , Sementes/química , Soro , Crânio/citologiaRESUMO
OBJECTIVE: To study the best ultrasonic technology of the extraction of icariin of Hugu capsule. METHODS: Used the content of icariin as index, orthogonal experiment was carried out to investigate 4 influential factors as follows: the ultrasonic power (A), the ultrasonic frequency (B), the material fluid ratio (C), the time (D). RESULTS: The best extraction conditions were as follows: the ultrasonic power was 120 W, the ultrasonic frequency was 28 KHz, solid-liquid ratio of 1 : 35, the extraction time was 10 min. CONCLUSION: Optimization of extracting process is simple, quick and low energy consumption. Under these conditions, the extraction of icariin is 1.8 times higher than that of the traditional extraction method.
Assuntos
Medicamentos de Ervas Chinesas/química , Flavonoides/isolamento & purificação , Tecnologia Farmacêutica/métodos , Ultrassom , Cápsulas , Cromatografia Líquida de Alta Pressão , Combinação de Medicamentos , Medicamentos de Ervas Chinesas/isolamento & purificação , Flavonoides/análise , Plantas Medicinais/química , Solventes/química , Fatores de TempoRESUMO
OBJECTIVE: To study the effect of drug serum of prepared Radix Polygoni Multiflori on rat osteoblast (Ob) and its mechanism. METHODS: The animal serum was prepared by serum pharmacology means. The cells were getting by separating and inducing the SD neonatal rat skull bone. The proliferation and differentiation of Ob were detected by CCK-8, alkaline phosphatase (ALP) activity analysis. And RT-PCR method was used to determine the osteogenesis-related genes expression. RESULTS: Compared with control group, the groups with drug serum of prepared Radix Polygoni Mutiflori, 10%, 20% and 30% had an effect on promotion the proliferation significantly on Ob (P < 0.01). There was no concentration-related manner among groups. The 5% and 10% drug serum decreased ALP activity at the post-translation phase compared with control group, but higher doses (20% and 30%) did not have the same effect. However, drug serum of PR/MIN increased significantly osteogenesis-related genes (OC, ALP, Cbfalpha1) mRNA expression (P < 0.05). CONCLUSION: The drug serum of prepared Radix Polygoni Multiflori can stimulate osteoblast proliferation in vitro, and its mechanism may be associated with increasing osteogenesis-related genes expression.
Assuntos
Fosfatase Alcalina/metabolismo , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Osteoblastos/efeitos dos fármacos , Polygonaceae/química , Fosfatase Alcalina/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Fatores de Ligação ao Core/genética , Fatores de Ligação ao Core/metabolismo , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Raízes de Plantas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soro , Crânio/citologiaRESUMO
Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(2)-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH(2)- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.
Assuntos
GMP Cíclico/análogos & derivados , Canais Epiteliais de Sódio/metabolismo , Oócitos/metabolismo , Tionucleotídeos/administração & dosagem , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , GMP Cíclico/administração & dosagem , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Condutividade Elétrica , Ativadores de Enzimas/farmacologia , Feminino , Humanos , Isoenzimas/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Lítio/farmacologia , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Fosforilação , Potássio/farmacologia , Isoformas de Proteínas , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/farmacologia , Ratos , Tionucleotídeos/farmacologia , Regulação para Cima , Xenopus laevisRESUMO
Correction for 'Development of a bidirectional isothermal amplification strategy for the sensitive detection of transcription factors in cancer cells' by Yan Zhang et al., Chem. Commun., 2020, 56, 8952-8955, DOI: 10.1039/D0CC03134H.
RESUMO
We developed a new strategy to sensitively detect transcription factors (TFs) based on the integration of a bidirectional isothermal exponential amplification reaction (EXPAR) with endonuclease IV (endo IV)-assisted cycle digestion of signal probes. This assay exhibits ultrahigh sensitivity with a detection limit of 1.29 × 10-14 M, and it can measure endogenous NF-κB p50 in HeLa cell extracts. Moreover, this strategy can be applied to screen TF inhibitors and detect other TFs by simply changing the TF-binding sequence.
Assuntos
Subunidade p50 de NF-kappa B/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Desoxirribonuclease IV (Fago T4-Induzido)/metabolismo , Células HeLa , Humanos , Limite de Detecção , Espectrometria de FluorescênciaRESUMO
OBJECTIVE: To investigate the effect of Rosiglitazone on the proliferation and the production of transforming growth factor-beta 1 (TGF-beta 1) in cultured humans Tenon's capsule fibroblasts (HTFs). METHODS: It was a experimental study. HTFs were cultured and subcultured in vitro. After HTFs were treated with different concentrations of Rosiglitazone, the proliferation was detected by MTT assay. The migration ability was detected by a scratch method. The production of TGF-beta 1 was determined by ELISA. TGF-beta 1 mRNA expression was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared with control group, Rosiglitazone significantly (F = 178.293, P < 0.01) inhibited the proliferation (0.33 +/- 0.01 vs 0.29 +/- 0.02) and the migration of HTFs in a dose-dependent manner with the concentrations ranged from 20 to 500 mg/L. Rosiglitazone also significantly (F = 86.404, P < 0.01) reduced the production of TGF-beta 1 in HTFs (323.48 +/- 13.69 vs 267.48 +/- 20.31). TGF-beta 1 mRNA was significantly (F = 176.102, P < 0.01) down-regulated (0.69 +/- 0.02 vs 0.55 +/- 0.02). CONCLUSIONS: This study suggests that the inhibition of the proliferation of Tenon's capsule and the migration of HTFs by Rosiglitazone is due, at least in part, to decrease the production of TGF-beta 1.
Assuntos
Olho/efeitos dos fármacos , Olho/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Tiazolidinedionas/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Olho/citologia , Humanos , RosiglitazonaRESUMO
We develop a label-free and high-throughput bioluminescence method for the sensitive detection of uracil DNA glycosylase (UDG) through enzyme-mediated tricyclic cascade signal amplification. This method exhibits high sensitivity with a detection limit as low as 0.00031 U mL-1, and it can be further applied for the measurement of enzyme kinetic parameters and the screening of UDG inhibitors as well as cancer cell analysis.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Medições Luminescentes/métodos , Uracila-DNA Glicosidase/análise , Neoplasias do Colo do Útero/enzimologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Feminino , Células HeLa , Humanos , Cinética , Uracila-DNA Glicosidase/antagonistas & inibidores , Uracila-DNA Glicosidase/metabolismo , Neoplasias do Colo do Útero/diagnósticoRESUMO
OBJECTIVE: To study the effect of aldosterone on cell proliferation, alkaline phosphatase (AKP) activity and osteogenic gene expression in rat osteoblasts and explore the mechanisms. METHODS: Osteoblasts isolated from the skull of neonatal SD rats by enzyme digestion were cultured and treated with different concentrations of aldosterone. The cell proliferation and AKP activity were evaluated using CCK-8 assay kit and AKP assay kit, respectively. The effects of aldosterone on mRNA and protein expressions of the osteogenic genes and epithelial sodium channel (ENaC) gene were investigated using semi-quantitative PCR and Western blotting. RESULTS: Compared with the control cells, the cells treated with 0.01-1.0 µmol/L aldosterone showed obviously enhanced proliferation while lower (1×10-3 µmol/L) or higher (10 µmol/L) concentrations of aldosterone did not significantly affect the cell proliferation. Aldosterone within the concentration range of 1×10-3 to 10 µmol/L did not cause significant changes in AKP activity in the osteoblasts. Treatment with 0.01 to 1.0 µmol/L aldosterone significantly upregulated the expressions of the osteogenic genes and α-ENaC gene at both the mRNA and protein levels. CONCLUSION: Aldosterone within the concentration range of 0.01-1.0 µmol/L stimulates the proliferation and osteogenic gene expressions and enhances α-ENaC gene expression in rat osteoblasts in vitro, suggesting the possibility that ENaC participates in aldosterone-mediated regulation of osteoblast functions.
Assuntos
Aldosterona/farmacologia , Fosfatase Alcalina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/genética , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Canais Epiteliais de Sódio/genética , Expressão Gênica , Osteoblastos/citologia , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To explore the role of epithelial sodium channel (ENaC) in regulating the functional activity of osteoclasts. METHODS: Multinucleated osteoclasts were obtained by inducing the differentiation of rat bone marrow cells with macrophage colony-stimulating factor (M-CSF) and RANKL. The osteoclasts were exposed to different concentrations of the ENaC inhibitor amiloride, and the expression of ENaC on osteoclasts was examined using immunofluorescence technique. The osteoclasts were identified with tartrate-resistant acid phosphatase (TRAP) staining, and the positive cells were incubated with fresh bovine femoral bone slices and the number of bone absorption pits was counted by computer-aided image processing. RT-PCR was performed to analyze the expression of cathepsin K in the osteoclasts. RESULTS: s Exposure to different concentrations of amiloride significantly inhibited the expression of ENaC and reduced the number of TRAP-positive osteoclasts. Exposure of the osteoclasts to amiloride also reduced the number of bone resorption pits on bone slices and the expression of osteoclast-specific gene cathepsin K. CONCLUSION: s ENaC may participate in the regulation of osteoclast differentiation and bone resorption, suggesting its role in functional regulation of the osteoclasts and a possibly new signaling pathway related with ENaC regulation for modulating bone metabolism.
Assuntos
Reabsorção Óssea , Canais Epiteliais de Sódio/metabolismo , Osteoclastos/citologia , Animais , Células da Medula Óssea/citologia , Catepsina K/metabolismo , Bovinos , Diferenciação Celular , Fator Estimulador de Colônias de Macrófagos/metabolismo , Ligante RANK/metabolismo , Ratos , Transdução de SinaisRESUMO
AIM: To determine the effect of piperazinyl estrone, a new estrogen derivative, on bone turnover, bone mass and uteri in ovariectomized rats. METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) or sham operated (sham) at the age of 3 months and treated with estrone (E) at 0.75 mg.kg-1.d-1, or with piperazinyl estrone (P-E) at 1 or 10 mg.kg-1.d-1, orally, for 3 months. At the time of death, the uterine weight was measured. Bone histomorphometric analysis of proximal tibial metaphyses (PTM) was performed in undecalcified sections. RESULTS: Bone histomorphometric data showed that the percent trabecular area (% Tb.Ar) of OVX rats with bone high turnover was significantly decreased. The uteri were atrophied. The percent trabecular area (% Tb.Ar) of estrone treated group was increased in decreasing bone turnover manner. But the size and weight of uteri in this group were increased vs OVX group. The bone loss induced by OVX was preserved by P-E treatment, but the mechanism of maintaining bone is different from that of E-treated rats. P-E treatment in low dose did not decrease any bone formation indices, such as percent labeling perimeter, bone formation rate per bone volume (BFR/BV), except bone mineral apposition rate (MAR) compared with E-treated group, and maintained them at OVX level. The uteri were found to be in atrophy compared with the match dose (0.75 mg) of E-treated OVX rats. But rats treated with high dose of P-E showed the same change like E-treated group. CONCLUSION: The finding of this study shows that lower dosage of piperazinyl estrone has effect on preventing the bone losses in OVX rats, while the bone formation and the uterus are not affected, thus supporting the hypothesis that piperazinyl estrone has the potential to prevent postmenopausal bone loss in women with less side effects.
Assuntos
Congêneres do Estradiol/uso terapêutico , Estrona/uso terapêutico , Osteogênese/efeitos dos fármacos , Osteoporose/prevenção & controle , Animais , Atrofia/prevenção & controle , Densidade Óssea , Congêneres do Estradiol/farmacologia , Estrona/análogos & derivados , Estrona/farmacologia , Feminino , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Ratos , Ratos Sprague-Dawley , Útero/patologiaRESUMO
AIM: To determine whether low dose of estrogen in combination with ginsenosides can completely prevent bone loss in ovariectomized rats. METHODS: Four-month-old ovariectomized rats were treated either with 100 and 300 mg.kg-1 of ginsenosides or 30 and 100 micrograms.kg-1 of 17 alpha-ethynylestradiol alone, or ginsenosides 100 mg.kg-1 in combination with 17 alpha-ethynylestradiol 30 micrograms.kg-1 for 10 weeks. Double in vivo fluorochrome labeling was made. The undecalcified longitudinal proximal tibial metaphyseal sections were processed and stained with Goldner's trichrome for histomorphometric analysis of the bone. RESULTS: Body weights and serum cholesterol were increased in ovariectomized (OVX) rats. The rats lost 74% of bone volume and high bone turnover was induced after OVX compared with the sham group. Bone volume increased by 205% in the high dose estrogen treated group while it was increased by 105% in the low dose group. The two doses of estrogen were shown to inhibit osteoclasts surface (by -65% and -55%, P < 0.01) and decrease bone turnover rate (by -85% and -83%, P < 0.01). High dose of estrogen was found to inhibit growth and stimulate uterine weight gain in rats while low dose did not. High dose of ginsenosides increased bone volume by 84% (P < 0.01) and decreased bone turnover rate by -64% (P < 0.05) while lower dose of ginsenosides did not. However, low dose ginsenosides combined with low dose estrogen achieved well preventive effects: increase of 202% in bone volume, decrease of 66% in bone turnover rate and 72% in osteoclasts surface. The combined effect in preventing bone loss equals to that the high dose of estrogen alone did. CONCLUSION: Use of low dose of estrogen plus ginsenosides showed synergistic effect on prevention of osteoporosis induced by ovariectomy.
Assuntos
Reabsorção Óssea/prevenção & controle , Etinilestradiol/uso terapêutico , Ginsenosídeos/uso terapêutico , Osteoporose/prevenção & controle , Animais , Densidade Óssea , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Etinilestradiol/administração & dosagem , Feminino , Ginsenosídeos/isolamento & purificação , Osteoporose/patologia , Ovariectomia , Panax/química , Folhas de Planta/química , Caules de Planta/química , Plantas Medicinais/química , Distribuição Aleatória , Ratos , Tíbia/patologiaRESUMO
OBJECTIVE: To evaluate the effects of stanozolol on the bone mineral density (BMD) and bone biomechanical properties of rats with glucocorticoid (GC)-induced osteoporosis (OP). METHODS: Twenty-eight male Sprague-Dawley rats of 3-month old were randomly divided into Group A (the basal control group), Group B (the age-matched control group), Group C (GC-induced OP group) and Group D (stanozolol-administrated group), 7 in each group. The rats in Group A were killed when experiment commenced, and those in Group B were given normal saline ig., while those in Groups C and D received the prednisone acetate (4.5 mg/kg, twice a week) alone and in combination with stanozolol (0.5 mg/kg, 6 times a week), respectively. Ninety days later, the bilateral femur and the 5th lumbar vertebra of the rats were isolated for BMD test using dual-energy X-ray absorptiometry scanner, and the torsion test, three-point bending test and compression test using electronic testing device. RESULTS: Compared with Group B, the mean BMD of the femur and the 5th lumbar vertebra in Group C decreased by 14.64% (P<0.01), the BMD of the bilateral distal femoral segment and the 5th lumbar vertebra decreased by 21.42% (P<0.01), 19.62% (P<0.05) and 23.48% (P<0.01) respectively. The load that the femur withstood in three-point bending test decreased by 17.1% (P<0.05), and the other biomechanical parameters also declined. When compared with Group C, the BMD in Group D increased, the torsional angle of the femur increased by 72.5% (P<0.05) and the other biomechanical parameters also tended to increase. CONCLUSIONS: BMD and biomechanical properties of the rat femur and the 5th lumbar vertebra decrease in response to a long-term GC administration, which can be prevented by stanozolol.