AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background: Activated hepatic stellate cells (HSCs) are closely involved in the initiation, perpetuation, andresolution of liver fibrosis. Pro-inflammatory cytokine levels are positively correlated with the transition from liverinjury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis. Methods: In this study, we investigatedthe effect of the pro-inflammatory cytokine interleukin (IL)-1β on the proliferation and signaling pathways involvedin fibrogenesis in LX-2 cells, an HSC cell line, using western blotting and cell proliferation assays. Results: IL-1βincreased the proliferation rate and α-smooth muscle actin (SMA) expression of LX-2 cells in a dose-dependentmanner. Within 1 h after IL-1β treatment, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB)signaling was activated in LX-2 cells. Subsequently, protein kinase B (AKT) phosphorylation and an increase in α-SMA expression were observed in LX-2 cells. Each inhibitor of JNK, p38, or NF-κB decreased cell proliferation, AKTphosphorylation, and α-SMA expression in IL-1β-treated LX-2 cells. Conclusion: These results indicate that JNK,p38, and NF-κB signals converge at AKT phosphorylation, leading to LX-2 activation by IL-1β. Therefore, the AKTsignaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.     

Human β-defensin 2 enhances IL-1β production and pyroptosis through P2X7-mediated NLRP3 expression in macrophages

Periodontal disease is the leading cause of tooth loss, which is also a high-risk factor for other diseases including oral cancer and cardiovascular disease. Periodontitis is one of the most common type of periodontal diseases. Interleukin-1β (IL-1β) plays a key role in the pathogenesis of periodontitis. However, the mechanism how IL-1β is produced during periodontitis is still unclear. In the present study, we found that human β-defensin 2 (hBD2) enhances IL-1β production through an LPS-primed human acute monocytic leukemia (THP-1) macrophage model. Inhibition of P2X purinoceptor 7 (P2X7) reduced hBD2-enhanced IL-1β production. Incubation of LPS-primed THP-1 macrophages with potassium chloride also suppressed hBD2-enhanced IL-1β production. Silence of inflammasome adaptor Nod-like receptor family pyrin domain containing 3 (NLRP3) led to reduced hBD2-enhanced IL-1β production. Likewise, inhibition of caspase-1 also resulted in the decrease of IL-1β. Moreover, an ethidium bromide uptake test indicated that hBD2-activated caspase-1 mediated pyroptotic pore formation. Subsequent lactate dehydrogenase detection and flow cytometric analysis indicated that hBD2 also induced pyroptosis. In brief, these findings illustrated not only the mechanism of hBD2 in enhancing the inflammatory response, but also provided novel therapeutic targets for periodontitis.     

Anti-inflammatory and antioxidant potential capacities of AD-MSCs and BM-MSCs in suppressing pancreatic β-cells auto-immunity and apoptosis in rats with T1DM induced model

Since Type 1 diabetes (T1DM) occurs when β-cells mass is reduced to less than 20% of the normal level due to autoimmune destruction of cells resulting in the inability to secrete insulin, preservation or replenishment of the functional β-cells mass has become a major therapeutic focus for this diabetic type treatment. Thus, this 4-week work plan was designed to determine which mesenchymal stem cells (MSCs) type is more appropriate to alleviate pancreatic hazards resulting from diabetes induction; via tracking a comparative study between MSCs derived from adipose tissue (AD-MSCs) and from bone marrow (BM-MSCs) in management of T1DM considering their immunomodulatory, anti-apoptotic and antioxidative roles. Rats were divided randomly into 4 groups; control, STZ-diabetic (D), D+AD-MSCs, and D+BM-MSCs groups. Both stem cells types in this study were allogenic. Herein, both oxidative stress and antioxidant markers were evaluated using colorimetric analysis, while inflammatory, immune and apoptotic markers were assessed through flow cytometric analysis. Results showed that diabetic rats treated with either AD-MSCs or BM-MSCs exhibited marked pancreatic antioxidant and anti-inflammatory activities that were able to initiate pancreatic immunomodulation and reducing β-cells apoptotic death, thus, help to restore their normal insulin secretion and hypoglycemic abilities. However, AD-MSCs injection was shown to be superior as a pancreatic regenerative tool in overcoming diabetes; owing to their marked antioxidant, anti-inflammatory, immunomodulatory, and anti-apoptotic characteristics over BM-MSCs treatment.     

F‐actin and α‐actinin reorganization mediates initial fibroblast interaction with CoCr alloy particles in vitro

Asceptic loosening remains the primary cause for failure of joint implant. The active role of fibroblasts in mediating asceptic loosening is however not well documented. In this study the initial interactions of fibroblasts with metal particles was studied by evaluating changes in the cytoskeletal structure and cytokine level. Murine L929 fibroblasts cultured with cobalt chromium particles were observed by phase contrast and scanning electron microscopy (SEM). Changes in the cytoskeletal rearrangement of F‐actin and α‐actinin focal adhesion plaques were studied by confocal microscopy. Expression of the proinflammatory cytokines IL‐6 and IL‐1α were analyzed by ELISA. The role of actin filaments and microtubules in particle uptake were determined at low temperature and in presence of colchicine and cytochalasin B. Phase contrast and SEM studies reveal that the metal particles adhere to the fibroblasts. The cellular cytoplasm was observed to grow over the particles and is suggestive of particle uptake. Confocal microscopy shows the presence of voids within the F‐actin cytoskeletal framework corresponding to areas occupied by the metal particles, indicating the possible uptake of these particles. Aggregates of α‐actinin into patches at the cell surface were also noted. Adherence and uptake of particles did not occur at low temperature and in presence of cytochalasin B, indicating that it is an active energy‐dependent process involving actin filaments. Changes in the levels of cytokine IL‐6 and IL‐1α were not observed suggesting the role of other cytokine molecules in mediating the inflammatory response to wear debri by fibroblasts. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Bushen Yizhi Formula regulates the IRE1α pathway to alleviate endoplasmic reticulum stress in an Alzheimer’s disease rat model

Background: While the Bushen Yizhi Formula can treat Alzheimer’s disease (AD), the yet to be ascertainedspecific mechanism of action was explored in this work. Methods: Different concentrations of the Bushen YizhiFormula and amyloid-beta peptide (Aβ) were used to treat rat pheochromocytoma cells (P12) and humanneuroblastoma cells (SH-SY5Y). Cell morphological changes were observed to determine the in vitro cell damage. CellCounting Kit (CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle,respectively. Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmicreticulum stress (ERS)-related proteins (GRP78 and CHOP), p-IRE1α, IRE1α, ASK1, p-JNK, JNK, Bax, Bcl-2, XBP-1,and Bim. Fura 2-acetoxymethyl ester (Fura-2/AM) was used to determine the intracellular calcium (Ca²⁺)concentration. Also, an AD model was constructed by injecting Aβ into the CA1 area of the hippocampus in SpragueDawley rats. AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutivedays. The Morris water maze experiment was conducted to test the learning and memory of rats. Hematoxylin &Eosin (H&E) and Terminal-deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL)staining were done to determine histopathological changes in the brain. Results: Bushen Yizhi Formula relieved theAβ-induced effects including cell injury, decreased viability, increased apoptosis, G0/G1 phase cell cycle arrest, upregulation of GRP78, CHOP, p-IRE1α, p-JNK, Bax, XBP-1 and Bim, as well as down-regulation of Bcl-2. Theseresults were also seen with IRE1α silencing. While Aβ suppressed the learning and memory abilities of rats, theBushen Yizhi Formula alleviated these effects of Aβ. Brain nerve cell injury induced by Aβ could also be treated withBushen Yizhi Formula. Conclusion: Bushen Yizhi Formula could influence ERS through the IRE1α signaling pathwayto achieve its therapeutic effects on AD.     

Ang-(1-7) exerts anti-inflammatory and antioxidant activities on high glucose-induced injury by prohibiting NF-κB-IL-1β and activating HO-1 pathways in HUVECs

Previous reports have suggested that Ang-(1-7) may have a protective effect in endothelial cells against highglucose (HG)-induced cell injury thanks to a modulatory mechanism in the NF-κB signaling pathway. In this study,we have examined whether NF-κB-IL-1β and Heme oxygenase-1 (HO-1) pathways contribute to the protection ofAng-(1-7) against hyperglycemia-induced inflammation and oxidative stress in human umbilical vein endothelial cells(HUVECs). Our results indicate that time-varying exposures of HUVECs, from 1 h to 24 h, to high glucoseconcentrations result in an increased expression of phosphorylated (p)-p65 and HO-1 in a time-dependent manner.As an inhibitor of NF-κB, pyrrolidinedithiocarbamic acid (PDTC) suppressed IL-1β production induced by HG. Ofnote, HUVECs previously treated with Ang-(1-7) (2 μM) for 30 min before being exposed to HG concentrationssignificantly ameliorated the HG-increased in p-p65 and IL-1β expression; whereas obviously up-regulated the level ofHO-1, along with inhibition of oxidative stress, inflammation, and the HG-induced cytotoxicity. Importantly, whenHUVECs were previously treated either with PDTC or IL-1Ra for 30 min before being exposed to HG, it significantlyprevented damages caused by high glucose concentrations mentioned above, while the treatment of HO-1 inhibitorSn-protoporphyrin (SnPP) before exposure to both HG and Ang-(1-7) significantly blocked the protective effectexerted by Ang-(1-7) on endothelial cells against injuries induced by HG mentioned above. To conclude, the data ofthis study showed that activation and inhibition of the NF-κB-IL-1β pathway and HO-1 pathway may constitute animportant defense mechanism against endothelial cell damage caused by HG concentrations. We additionally gavenew evidence showing that exogenous Ang-(1-7) exerts a protective effect on HUVECs against the HG-induced cellinjury via the inhibition and the activation of the NF-κB-IL-1β pathway and the HO-1 pathway, respectively.     

Differential expression and regulation of PNA and UEA‐1 bindings in rabbit uterus during preimplantation period

Peanut agglutinin (PNA) and Ulex europaeus agglutinin‐1 (UEA‐1) were used as probes to study the distribution of β‐gal (1→3) ga1Nac and α‐l ‐Fucose in rabbit uterus during early pregnancy. PNA binding was mainly localized on the surface of uterine glandular and luminal epithelium. There were no positive signals on day 1 of pregnancy. PNA binding gradually increased from day 2 and reached its highest level on days 3 and 4. The distribution of PNA binding gradually declined from day 5 and reached a low level on day 7. However, UEA‐1 binding was only localized on the luminal epithelial during early pregnancy. A high level of UEA‐1 binding had been found on the luminal epithelium on day 1 of pregnancy and low level of positive signals had been found in the uterus on days 2 and 3. UEA‐1 binding increased gradually and reached its highest level on day 4. Then the distribution of UEA‐1 binding sharply declined and no positive signals were found on days 5–7. The distribution of PNA and UEA‐1 bindings in pseudopregnant uterus was similar to that in normal pregnant uterus. During estrus cycle, there was no detectable PNA binding signal in uterus. But, a high level of UEA‐1 binding was found in the luminal epithelium of estrus uterus. In ovariectomized rabbit uterus, progesterone significantly induced the expression of PNA binding, while estrogen stimulated UEA‐1 binding expression. These results suggested that the distribution of PNA and UEA‐1 bindings in rabbit uterus may be related to rabbit implantation. Microsc. Res. Tech. 76:398–403, 2013. © 2013 Wiley Periodicals, Inc.     

Micro‐community cytometry: sensing changes in cell health and glycoconjugate expression by imaging and flow cytometry

Discerning the extent of biologically relevant heterogeneity presents unique challenges to both microscopy and flow cytometry. Micro‐environmental influences and stochastic changes in cellular behaviour can act to mask the origins of both progression and therapeutic resistance in tumour cell systems. In part the dimensionality of different and frequently metastable states can be assessed by multi‐parameter flow cytometry with unparalleled statistical robustness. Complementary application of imaging can provide valuable insights into the complex temporal changes that can occur in cell micro‐communities either spontaneously or in response to selection pressure. With an extensive range of methodologies for the labelling of cells there are multiple options for tracking cells, defining fate and the re‐construction of provenance and behavioural history. The challenge is highlighted by attempts to identify the critical glycosylation events modifying the function of cell surface proteins. Central to a cytometric approach is the availability of methods that reveal cell health and are compatible with the detection of cell surface changes within dynamic micro‐communities. The review briefly addresses the options for sensing cell health and the co‐application of an antibody mimetic for detection of cell surface glycoconjugate expression accessible for both imaging and flow cytometry.     

Overexpression of a sugarcane<i> ScCaM</i> gene negatively regulates salinity and drought stress responses in transgenic <i>Arabidopsis thaliana</i>

Calmodulin (CaM) proteins play a key role in signal transduction under various stresses. In the present study,the effects of a sugarcane ScCaM gene (NCBI accession number: GQ246454) on drought and salt stress tolerance intransgenic Arabidopsis thaliana and Escherichia coli cells were evaluated. The results demonstrated a significantnegative role of ScCaM in the drought and salt stress tolerance of transgenic lines of A. thaliana, as indicated by thephenotypes. In addition, the expression of AtP5CS and AtRD29A, two genes tightly related to stress resistance, wassignificantly lower in the overexpression lines than in the wild type. The growth of E. coli BL21 cells expressingScCaM showed weaker tolerance under mannitol and NaCl stress. Taken together, this study revealed that the ScCaMgene plays a negative regulatory role in both mannitol and NaCl stresses, and it possibly exerts protective mechanismscommon in both prokaryotes and eukaryotes under stress conditions.     

Computer simulation of turbine oil oxidation. 1: Consumption of a hindered phenol antioxidant in model hydrocarbon systems at 115°C

Computer simulation of the inhibited oxidation of model hydrocarbon systems representing turbine oils of different composition, hexadecane/tetralin mixtures containing di-tert-butyl pcresol (DBPC), has been achieved using the kinetic and mechanistic information obtained from fundamental hydrocarbon autoxidation and inhibition studies. The oxidation life, the time to complete consumption of DBPC and its reactive intermediates, was calculated using previously reported and newly measured absolute rate constants for reaction involved. The results of this calculation of oxidation life were in good agreement with values experimentally determined. The oxidation life has been found to decrease with increasing amount of tetralin in the mixtures. This can be explained by the increased rate of consumption of antioxidants, caused by an increase in the rate of free radical formation, via the direct reaction of hydrocarbons with oxygen, and via the homolytic decomposition of hydroperoxides. The observed decrease in oxidation life, however, is not significant, despite the fact that the addition of tetralin considerably increases the rate of free-radical formation. The relatively low sensitivity of oxidation life to the rate of free-radical formation caused by the addition of an easily oxidisable substrate can be attributed to the occurrence of a direct oxidation of antioxidants. That is, kinetic analysis for the consumption of antioxidants disclosed that antioxidant consumption mostly occurred not through the inhibition of oxidation, but through direct oxidation. The direct oxidation of antioxidants obeys the rate equation -d[AH]/dt = k₆[AH]^1.0[O₂]^1.5 where k₆ can be calculated, at various temperatures, from Arrhenius parameters obtained in this study (log (A/M⁻¹s⁻¹) = 9.6 and Ea = 22.6 kcall mol).     

Overexpression of inhibin α (1-32) fusion protein promotes apoptosis and cell cycle arrest in a cervical cancer cell model (Hela cells)

Inhibins play important roles in the reproductive system. To evaluate whether inhibin α (1-32) fusion protein plays a role in cervical cancer growth, the plasmid pVAX-inhα was constructed and its effect on proliferation and apoptosis of the human cervical cancer cell line (Hela) was checked by flow cytometry and real-time PCR. The expression and localization of inhibin α protein were detected by RT-PCR and confocal microscopy which showed that inhibin α protein was expressed and localized in the nucleus of Hela cells. Over expression of inhibin α gene significantly induced cell apoptosis and ceased S phase of cell cycle. Furthermore, cell proliferation was significantly suppressed 96 h post-transfection and mRNA level of anti-apoptosis genes (Bcl-2, NFκB) were decreased but pro-apoptosis genes (Bax, wild type p53) and inhibin co receptor (TGFβR3) were increased, indicating that inhibin, through its co-receptor, might activate apoptotic and cell growth cascades which regulate proliferation and apoptosis in Hela cells. These results suggest that inhibin α (1-32) fusion protein, located in the cell nucleus, can regulate Hela cells growth and apoptosis by induction of apoptotic pathways such as NFκB, Bcl-2 and p53 families. These findings may have a significant impact on future research regarding cervical cancer cell lines     

Mechanical and histological properties of an electrospun scaffold with a modified surface by plasma polymerization implanted in an <i>in vivo</i> model

This article presents the construction of scaffolds composed of polylactic acid (PLA) with different concentrations of hydroxyapatite (HA) by electrospinning, which were superficially modified with polypyrrole (PPy/I) by plasma polymerization. A preliminary study was conducted of the biological and mechanical behavior of the scaffolds when they were implanted in the back of rabbits for 30 days; bone cells differentiated from mesenchymal stem cells (MSCs) were used. The bone cell and scaffold structures were characterized by histological, immunohistochemical, and mechanical stress tests. Hematoxylin–eosin staining showed good tissue conformation. The immunohistochemical tests highlighted the presence of the main bone tissue proteins, such as collagen, osteocalcin, and osteopontin. The PLA/HA scaffolds were observed to exhibit cell adhesion and proliferation properties; however, the response was much better in the scaffolds that had a higher concentration of HA and that were coated with PPy/I. The results of the mechanical tests of the scaffolds indicated that the plasma treatment improved the adhesion and cell proliferation properties and contributed to the mechanical support, allowing the formation of neotissues with good viability of cell growth.     

Evaluation of actual sensitivity limit in a 10 N·m dead weight torque standard machine and stability of a new 1 N·m torque transducer

A 10 N·m dead weight torque standard machine (10-N·m-DWTSM) has been under development at NMIJ/AIST since 2006 to expand the range of the torque standard. Estimation of the sensitivity limit of the fulcrum is one of the most important issues to realize a precise reference torque of small capacity by using a dead weight torque standard machine. In this study, a torque transducer was installed on the 10-N·m-DWTSM in order to keep the moment-arm on the horizontal line (balancing). The sensitivity limit of the fulcrum under real calibration conditions was estimated by reading the change in the output from the torque measuring device (TMD: the torque transducer with a cable and an indicating device) when small weights were loaded or unloaded. The small weights used in the experiment were 0.5 mg, 1 mg, 10 mg, and 100 mg. Equivalent radial loads from 0.1 N·m to 10 N·m were imposed on the fulcrum during the sensitivity measurement.     

An orientation‐independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation‐independent differential interference contrast microscope for in vivo imaging of wound healing. Orientation‐independent differential interference contrast provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, nontransgenic animal model. In particular, the orientation‐independent differential interference contrast microscope equipped with a 40x/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized.     

Comparison of an ultrasonic interferometer manometer and a new dynamic flow control system in the pressure range 1–133 Pa

This work is concerned with comparison of two low-pressure calibration systems at the Korea Research Institute of Standards and Science (KRISS). The ultrasonic interferometer manometer (UIM) which is a national primary standard and the dynamic flow control system (FCS) which can be used for the calibration of vacuum gauges by comparison method by generating precise and stable pressure in the range 1–133 Pa. For this comparison, a capacitance diaphragm gauge (CDG) of 133 Pa full-scale range was used as transfer standard in the overlapping range of 1–133 Pa. The behavior of two systems in the pressure range 1.37–11.5 Pa had large variations which gradually reduced above 11.5 Pa. In the range 1.37–11.5 Pa, the difference in calibration factors for the two systems was 0.0114 with relative deviations of 5.177%. However above 11.5 Pa the corresponding values were 0.0056 and 1.197%, respectively. Since in vacuum metrology, UIM is world-wide recognized as a national calibration standard, the results of this comparison will be used to validate the FCS for calibration of low vacuum gauges.     

Microstructural evolution of Cu-1 at% Ti alloy aged in a hydrogen atmosphere and its relation with the electrical conductivity

Copper alloys with titanium additions between 1 and 6 at% Ti emerge currently as attractive conductive materials for electrical and electronic commercial products, since they exhibit superior mechanical and electrical properties. However, their electrical conductivity is reduced owing to the residual amount of Ti solutes in the Cu solid solution (Cu_ss) phase. Since Cu shows only poor reactivity with hydrogen (H), while Ti exhibits high affinity to it, we were inspired by the idea that hydrogenation of Cu–Ti alloys would influence their microstructure, resulting in a significant change of their properties. In this contribution, the influence of aging under a deuterium (D₂) atmosphere of Cu-1 at% Ti alloys on their microstructure is investigated to explore the effects on the electrical conductivity. The specimens were investigated by means of transmission electron microscopy (TEM), field ion microscopy (FIM), computer-aided field ion image tomography (cFIIT), and atom probe tomography (APT).