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BACKGROUND: Hepatic fibrosis is a necessary step in the development of hepatic cirrhosis. In this study we used lentiviral vector-mediated transfection technology to evaluate the effect of peroxisome proliferator-activated receptor gamma (PPAR-gamma) on rat hepatic fibrosis. METHODS: Hepatic fibrosis in rats was induced by CCl4 for 2 weeks (early fibrosis) and 8 weeks (sustained fibrosis). The rats were randomly divided into four groups: normal control, fibrosis, blank vector, and PPAR-gamma. They were infected with the recombinant lentiviral expression vector carrying the rat PPAR-gamma gene by portal vein injection. The liver of the rats was examined histologically and hydroxyproline was assessed. In vitro primary hepatic stellate cells (HSCs) were infected with the recombinant lentiviral expression vector carrying the rat PPAR-gamma gene. The status of HSC proliferation was measured by the MTT assay. The protein levels of PPAR-gamma, alpha-smooth muscle actin (alpha-SMA) and type I collagen expression were evaluated by the Western blotting method. RESULTS: In vitro studies revealed that expression of PPAR-gamma inhibited expression of alpha-SMA and type I collagen in activated HSCs (P<0.01) as well as HSC proliferation (P<0.01). In vivo experiments indicated that in the early hepatic fibrosis group, the hydroxyproline content and the level of collagen I protein in the liver in the PPAR-gamma transfected group were not significantly different compared to the hepatic fibrosis group and the blank vector group; whereas the expressions of PPAR-gamma and alpha-SMA were different compared to the hepatic fibrosis group (P<0.01). In the sustained hepatic fibrosis group, there were significant differences in the hydroxyproline content and the expression of PPAR-gamma, alpha-SMA, and type I collagen between each group. CONCLUSION: PPAR-gamma can inhibit HSC proliferation and hepatic fibrosis, and suppress alpha-SMA and type I collagen expression.
Assuntos
Proliferação de Células , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Fígado/patologia , PPAR gama/metabolismo , Actinas/metabolismo , Animais , Tetracloreto de Carbono , Células Cultivadas , Colágeno Tipo I/metabolismo , Expressão Gênica , Hidroxiprolina/metabolismo , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Masculino , PPAR gama/genética , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
With the increasing application of enzymes in various research fields, the choices of co-solvents in enzymatic preparations which directly related to the catalytic activity have been attracted attention. Thus, researching on the stabilization or destabilization behaviors of enzymes in different solvents is extremely essential. In this study, the structural changes of DhaA in two typical aprotic co-solvents (acetonitrile and tetrahydrofuran) were firstly investigated by molecular dynamics (MD) simulation. The simulation results revealed the strong van der Waals force between co-solvents and DhaA which could induce the structural change of enzyme. Interestingly, the differences of molecular size and the electrostatic force with enzyme of two co-solvents led to quite different influences on DhaA. As for acetonitrile, solvent molecules could penetrate into the catalytic site of DhaA which promoted by the electrostatic interaction. On the contrary, tetrahydrofuran molecules were mainly distributed around the catalytic site due to the relative weak electrostatic interaction and steric resistance effect. It can be concluded that different co-solvent can affect the key domains, substrate pathway and catalytic pocket of DhaA.Communicated by Ramaswamy H. Sarma.
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Hidrolases , Simulação de Dinâmica Molecular , Catálise , Domínio Catalítico , SolventesRESUMO
Two-photon polymerization of a three-dimensional (3D) hydrogel structure has been widely applied in biological tissue engineering. For improving the biocompatibility of hydrogel structures, a new kind of ionic carbazole water-soluble photoinitiator was prepared to realize the fabrication of a 3D hydrogel structure in aqueous phase. 3,6-Bis[2-(1-methyl-pyridinium)vinyl]-9-methyl-carbazole diiodide (BMVMC) and cucurbit[7]uril (CB7) have been employed to generate a complex with better water solubility by host-guest interactions. The binding ratio of the complex was demonstrated to be 1:1 through the characterization of isothermal titration calorimetry (ITC). The two-photon absorption (TPA) cross section of the complex increases to 2500 GM compared with the 750 GM of the BMVMC molecule. Then, an aqueous-phase photoresist was obtained using the CB7/BMVMC complex as the photoinitiator and poly(ethylene glycol) diacrylate (PEGda) as the hydrogel monomer. Two-photon fabrication capability in aqueous phase has been studied using the as-prepared photoresist. A low laser threshold of 3.7 mW as well as a high resolution of 180 nm are achieved. Benefiting from the fluorescence properties of the photoinitiator, we can achieve the confocal fluorescence images without any assistance of fluorescent probes. Subsequently, a 3D engineered hydrogel scaffold microstructure was fabricated by the two-photon polymerization technology, whose biocompatibility was demonstrated by culturing the structure with living cells of L929. The BMVMC-CB7 complex and the as-prepared photoresist are demonstrated to have good biocompatibility, which is prospective for further application in tissue engineering.
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Carbazóis/química , Hidrogéis/química , Alicerces Teciduais/química , Animais , Hidrocarbonetos Aromáticos com Pontes/síntese química , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/toxicidade , Carbazóis/síntese química , Carbazóis/efeitos da radiação , Carbazóis/toxicidade , Linhagem Celular , Módulo de Elasticidade , Hidrogéis/síntese química , Hidrogéis/efeitos da radiação , Hidrogéis/toxicidade , Imidazóis/síntese química , Imidazóis/química , Imidazóis/toxicidade , Camundongos , Fótons , Polimerização/efeitos da radiação , Solubilidade , Engenharia Tecidual/métodos , Água/químicaRESUMO
The efficient immobilization of haloalkane dehalogenase (DhaA) on carriers with retaining of its catalytic activity is essential for its application in environmental remediation. In this work, adsorption orientation and conformation of DhaA on different functional surfaces were investigated by computer simulations; meanwhile, the mechanism of varying the catalytic activity was also probed. The corresponding experiments were then carried out to verify the simulation results. (The simulations of DhaA on SAMs provided parallel insights into DhaA adsorption in carriers. Then, the theory-guided experiments were carried out to screen the best surface functional groups for DhaA immobilization.) The electrostatic interaction was considered as the main impact factor for the regulation of enzyme orientation, conformation, and enzyme bioactivity during DhaA adsorption. The synergy of overall conformation, enzyme substrate tunnel structural parameters, and distance between catalytic active sites and surfaces codetermined the catalytic activity of DhaA. Specifically, it was found that the positively charged surface with suitable surface charge density was helpful for the adsorption of DhaA and retaining its conformation and catalytic activity and was favorable for higher enzymatic catalysis efficiency in haloalkane decomposition and environmental remediation. The neutral, negatively charged surfaces and positively charged surfaces with high surface charge density always caused relatively larger DhaA conformation change and decreased catalytic activity. This study develops a strategy using a combination of simulation and experiment, which can be essential for guiding the rational design of the functionalization of carriers for enzyme adsorption, and provides a practical tool to rationally screen functional groups for the optimization of adsorbed enzyme functions on carriers. More importantly, the strategy is general and can be applied to control behaviors of different enzymes on functional carrier materials.
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Hidrolases/química , Rhodococcus/enzimologia , Adsorção , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Hidrolases/metabolismo , Modelos Moleculares , Eletricidade Estática , Propriedades de SuperfícieRESUMO
We have introduced a novel water-soluble two-photon photoinitiator based on the host-guest interaction between 3,6-bis[2-(1-methyl-pyridinium)vinyl]-9-pentyl-carbazole diiodide (BMVPC) and cucurbit[7]uril (CB7) because most of the commercial photoinitiators have poor two-photon initiating efficiency in aqueous solutions. The binding ratio of BMVPC and CB7 was determined as 1:1 by isothermal titration calorimetry and quantum chemical calculation. The formation of the host-guest complex increases the two-photon absorption cross-section about five times, and improves the water solubility required as the photoinitiator for hydrogel fabrication. The BMVPC-CB7 inclusion complex was used as the one-component photoinitiator, and the polyethylene glycol diacrylate with promising biocompatibility was used as the hydrogel monomer to form the aqueous-phase photoresist system applied to two-photon polymerization microfabrication. A relatively low laser threshold of 4.5 mW, a high fabricating resolution of 180 nm, and the true three-dimensional (3D) fabricating capability in the aqueous solution have been obtained by using the as-prepared photoresist system. Finally, 3D engineering hydrogel scaffold microstructures with low toxicity and good biocompatibility have been fabricated and cocultured with living HeLa cells, which demonstrates the potential for further application in tissue engineering.
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Materiais Biocompatíveis , Hidrocarbonetos Aromáticos com Pontes/química , Carbazóis/química , Hidrogéis , Imidazóis/química , Lasers , Alicerces Teciduais/química , Materiais Biocompatíveis/química , Células HeLa , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Microtecnologia , Polietilenoglicóis/química , Engenharia TecidualRESUMO
In this study, two kinds of novel carbazole-based ethynylpyridine salts: 3,6-bis[2-(1-methylpyridinium)ethynyl]-9-pentyl-carbazole diiodide (BMEPC) and 3,6-bis[2-(1-methylpyridinium)ethynyl]-9-methyl-carbazole diiodide (BMEMC) have been employed as photosensitizers owing to their excellent antibacterial activity. These molecules possess symmetric A-π-D-π-A-type structures, which would bring in the unique optical properties. The inhibition zone measurement of a gradient concentration from 0 to 100 µM showed BMEPC and BMEMC photoinduced antibacterial activity against Escherichia coli. Diameters of zone of inhibition were up to 15 and 14 mm under laser irradiations. Under the exposure of the laser of 442 nm with a power density of 20 mW/cm2, the minimum inhibitory concentrations (MICs) of BMEPC on E. coli were between 3.5 and 6.9 µM and that of BMEMC were between 9.4 and 18.8 µM, respectively. In the dark experiments as a control, the MIC value is between 6.9 and 13.8 µM for BMEPC, whereas it is between 187.5 and 225.0 µM for BMEMC. By the comparison of the MIC values of BMEPC and BMEMC with laser irradiation and in dark, the laser-induced toxicity on bacteria is more evident, though both of the derivatives have dark toxicity. With the laser irradiation duration of 30 s and 10 min for BMEPC and BMEMC, respectively, the survival rate of E. coli approximates zero. An antibacterial mechanism has been proposed based on the electron paramagnetic resonance characterization, which indicates that a nitride radical is generated under laser irradiation. The carbazole-based ethynylpyridine photosensitizers would provide high potential for further applications in photodynamic therapy.
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Microenvironment in biology is diverse and complex which has been a great challenge for in vivo imaging materials, and so materials with environmental tolerance and photostability need to be explored. For aggregation-induced emission (AIE) molecules, the fluorescence is closely related to the restricted structure which is directly affected by the microenvironment. Inorganic silica nanoparticles can provide a rigid microenvironment which can stabilize AIE molecules to obtain fluorescent materials with environmental tolerance. Here, stable fluorescent SiO2 nanoparticles (CWQ-11@SiO2 NPs) have been prepared by doping with typical AIE molecules named CWQ-11. CWQ-11@SiO2 NPs have narrow size distribution and spherical morphology with a size of around 50 nm. The fluorescence intensity of CWQ-11@SiO2 NPs is nearly 45.4 times higher than that of free CWQ-11. CWQ-11@SiO2 NPs maintain excellent fluorescence stabilities under various conditions, such as in solutions with different pH values, different viscosities, or continuous irradiation, and even in simulated gastric fluid (SGF). Cellular imaging research represents efficient imaging ability of CWQ-11@SiO2 NPs in two different tumor cells including MCF-7S and HepG-2. All these results demonstrate that the CWQ-11@SiO2 NPs have been successfully prepared and remain stable under different harsh conditions, and have promising potential in imaging, tracing for drugs or diagnosis in complicated biological systems.
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Here, we have introduced a novel biscarbazolylmethane-based cyanine as a two-photon excited fluorescent probe, 6,6'-bis[2-(1-methylpyridinium)vinyl]-bis(9-methyl-carbazol-3-yl)methane diiodide, which has two vinylpyridinium carbazole moieties connected by a non-rigid methylene bridge. This molecule possesses a larger Stokes shift and enhanced two-photon absorption cross-section than the previously reported vinylpyridinium carbazole monocyanine, which is mainly attributed to the "through-space" type intramolecular charge transfer. The low fluorescence quantum yield and 30-fold fluorescence enhancement once binding with calf thymus DNA highlight this molecule as a promising fluorescence light-up probe for DNA. The obvious induced circular dichroism signals have proved that the molecule with soft-connected bis-cationic centers can specifically interact with various DNA structures. Cell viability study shows that the probe has very low cytotoxicity. The probe exhibits high staining selectivity for mitochondria in living HeLa cells. Its capability to stain nuclear DNA has been confirmed by fixed cell staining. Furthermore, the application for two-photon excited fluorescence imaging demonstrates high potential of the probe for nonlinear bioimaging with 3D resolution.