Pyroptosis in stroke-new insights into disease mechanisms and therapeutic strategies

Journal of Physiology and Biochemistry - Stroke is a common disease with high mortality and disability worldwide. Different forms of cell deaths, including apoptosis and necrosis, occur in ischemic...     

Genistein-3′-sodium sulfonate Attenuates Neuroinflammation in Stroke Rats by Down-Regulating Microglial M1 Polarization through α7nAChR-NF-κB Signaling Pathway

Microglial M1 depolarization mediated prolonged inflammation contributing to brain injury in ischemic stroke. Our previous study revealed that Genistein-3′-sodium sulfonate (GSS) exerted neuroprotective effects in ischemic stroke. This study aimed to explore whether GSS protected against brain injury in ischemic stroke by regulating microglial M1 depolarization and its underlying mechanisms. We established transient middle cerebral artery occlusion and reperfusion (tMCAO) model in rats and used lipopolysaccharide (LPS)-stimulated BV2 microglial cells as in vitro model. Our results showed that GSS treatment significantly reduced the brain infarcted volume and improved the neurological function in tMCAO rats. Meanwhile, GSS treatment also dramatically reduced microglia M1 depolarization and IL-1β level, reversed α7nAChR expression, and inhibited the activation of NF-κB signaling in the ischemic penumbra brain regions. These effects of GSS were further verified in LPS-induced M1 depolarization of BV2 cells. Furthermore, pretreatment of α7nAChR inhibitor (α-BTX) significantly restrained the neuroprotective effect of GSS treatment in tMCAO rats. α-BTX also blunted the regulating effects of GSS on neuroinflammation, M1 depolarization and NF-κB signaling activation. This study demonstrates that GSS protects against brain injury in ischemic stroke by reducing microglia M1 depolarization to suppress neuroinflammation in peri-infarcted brain regions through upregulating α7nAChR and thereby inhibition of NF-κB signaling. Our findings uncover a potential molecular mechanism for GSS treatment in ischemic stroke.     

脐带血造血干/祖细胞体外分化为不同阶段红系祖细胞的动力学观察

目的:探讨体外培养脐带血单个核细胞定向诱导分化为不同阶段红系祖细胞的动力学变化情况。方法:用0.5%甲基纤维素沉降脐带血红细胞及人淋巴细胞分离液密度梯度离心法得到单个核细胞,在含EPO、SCF、IGF-1等细胞因子的无血清培养体系中诱导其定向分化为红系祖细胞,观察细胞增殖、存活率、细胞集落形成情况,并检测不同阶段细胞红系特异性表面标志CD71和CD235a的表达。结果:随着培养时间的延长,细胞数逐渐增多,14 d细胞可扩增140倍左右,收集诱导后的细胞进行瑞氏吉姆萨染色,可见大量红系祖细胞,诱导后的细胞集落形成能力强,形成的克隆大部分为红系集落。诱导过程中,14 d前CD71、CD235a的表达逐渐增高。按细胞表面标志表达的不同可将诱导的细胞分为4群,分别对应红系祖细胞的不同阶段;随着诱导天数的增加,各时间点细胞对应的早期红系祖细胞群(P2、P3)比例逐渐下降,中晚期红系祖细胞群(P4、P5)的比例逐渐上升。结论:无血清培养基添加细胞因子组合的红系诱导培养体系可较好地诱导扩增红系祖细胞,流式分选可获得相对均一而处于不同分化阶段的红系祖细胞群体。获得了红系祖细胞体外分化的动力学数据,为今后进一步优化红系诱导分化体系获得均一的红系祖细胞奠定了基础,并对未来利用干细胞制备均一的红系祖细胞应用于临床治疗有一定的指导作用。     

Geometrical optimisation of a biochip microchannel fluidic separator

This article reports on the geometric optimisation of a T-shaped biochip microchannel fluidic separator aiming to maximise the separation efficiency of plasma from blood through the improvement of the unbalanced separation performance among different channel bifurcations. For this purpose, an algebraic analysis is firstly implemented to identify the key parameters affecting fluid separation. A numerical optimisation is then carried out to search the key parameters for improved separation performance of the biochip. Three parameters, the interval length between bifurcations, the main channel length from the outlet to the bifurcation region and the side channel geometry, are identified as the key characteristic sizes and defined as optimisation variables. A balanced flow rate ratio between the main and side channels, which is an indication of separation effectiveness, is defined as the objective. It is found that the degradation of the separation performance is caused by the unbalanced channel resistance ratio between the main and side channel routes from bifurcations to outlets. The effects of the three key parameters can be summarised as follows: (a) shortening the interval length between bifurcations moderately reduces the differences in the flow rate ratios; (b) extending the length of the main channel from the main outlet is effective for achieving a uniformity of flow rate ratio but ineffective in changing the velocity difference of the side channels and (c) decreasing the lengths of side channels from upstream to downstream is effective for both obtaining a uniform flow rate ratio and reducing the differences in the flow velocities between the side branch channels. An optimisation process combining the three parameters is suggested as this integration approach leads to fast convergent process and also offers flexible design options for satisfying different requirements.     

Integration of geometric separation mechanisms by implementing curved constrictions in a biochip microchannel fluidic separator

This paper investigates the effectiveness of using curved constrictions in the bifurcation region of T-type fluid separators for promoting flow development in the intervals between bifurcations. A design of biofluid separator is proposed and a mathematical analysis and a numerical simulation of the blood flow in microchannels are conducted. The design is based on a modification of an existing T-shaped biochip device which consists of a main channel and a series of perpendicularly positioned side channels. By means of bifurcation effect, the blood is separated into plasma concentration flow from the side channels and blood cell concentration flow from the main channel. In this design, curved constrictions are inserted between bifurcations to replace the original straight channel section, so that the constriction and curved channel effects can be induced apart from the existing bifurcation effect. The mathematical analysis is aimed to the flow field and shear stress of the blood fluid in the microchannel geometries employed in the current design, including bifurcation, constriction and curved channel. The numerical simulation and mathematical analysis result in agreed conclusions, giving some insights into the importance of the relevant geometries in promoting biofluid separation. The main results can be summarised as follows: (i) the constrictions can largely increase the shear stress by the ratio of square of the reduction of the sections between the constriction and parent main channel. (ii) The curved channel intervals can induce centrifugal force, smoothly transit the flow field and increase the chances depleting fluid from the cell-free layer. (iii) The thickness of the boundary layer skimmed into the side channels from the main channel is decreased in this design and can be controlled, falling into the cell-free layer region by adjusting the geometry of the side channels.     

Effect of membrane stiffness and cytoskeletal element density on mechanical stimuli within cells: an analysis of the consequences of ageing in cells

A finite element model of a single cell was created and used to compute the biophysical stimuli generated within a cell under mechanical loading. Major cellular components were incorporated in the model: the membrane, cytoplasm, nucleus, microtubules, actin filaments, intermediate filaments, nuclear lamina and chromatin. The model used multiple sets of tensegrity structures. Viscoelastic properties were assigned to the continuum components. To corroborate the model, a simulation of atomic force microscopy indentation was performed and results showed a force/indentation simulation with the range of experimental results. A parametric analysis of both increasing membrane stiffness (thereby modelling membrane peroxidation with age) and decreasing density of cytoskeletal elements (thereby modelling reduced actin density with age) was performed. Comparing normal and aged cells under indentation predicts that aged cells have a lower membrane area subjected to high strain as compared with young cells, but the difference, surprisingly, is very small and may not be measurable experimentally. Ageing is predicted to have a more significant effect on strain deep in the nucleus. These results show that computation of biophysical stimuli within cells are achievable with single-cell computational models; correspondence between computed and measured force/displacement behaviours provides a high-level validation of the model. Regarding the effect of ageing, the models suggest only small, although possibly physiologically significant, differences in internal biophysical stimuli between normal and aged cells.     

Design,synthesis and in vitro biological evaluation of quinazolinone derivatives as EGFR inhibitors for antitumor treatment

Abstract

In this paper, a series of novel 3-methyl-quinazolinone derivatives was designed, synthesised and evaluated for antitumor activity in vitro on wild type epidermal growth factor receptor tyrosine kinase (EGFR^wt-TK) and three human cancer cell lines including A549, PC-3, and SMMC-7721. The results displayed that some of the compounds had good activities, especially 2-{4-[(3-Fluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5?g), 2-{4-[(3,4-Difluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5k) and 2-{4-[(3,5-Difluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5?l) showed high antitumor activities against three cancer cell lines. Moreover, compound 5k could induce late apoptosis of A549 cells at high concentrations and arrest cell cycle of A549 cells in the G2/M phase at tested concentrations. Also, compound 5k could inhibit the EGFR^wt-TK with IC₅₀ value of 10?nM. Molecular docking data indicates that the compound 5k may exert inhibitory activity by forming stable hydrogen bonds with the R817, T830 amino acid residues and cation-Π interaction with the K72 residue of EGFR^wt-TK.     

Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.