Recombinant human elafin protects airway epithelium integrity during inflammation

Elafin, an antiprotease, is likely to protect pulmonary epithelial cells from inflammatory damages. We aimed to explore the molecule mechanisms of recombinant human elafin on protecting A549 cells integrity against inflammatory assault. We transfected A549 airway epithelial cells with eukaryotic expression vector pEGFP-N1-Elafin and negative control vector pEGFP-N1, respectively. Cells were co-incubated with polymorphonuclear neutrophils (PMN) and then were stimulated with lipopolysaccharide (LPS). Results revealed that, in pEGFP-N1-Elafin transfected cells, neutrophil elastase (NE) activity significantly decreased after LPS stimulation, accompanied with elevated elafin mRNA level and protein production, whereas in cells transfected with pEGFP-N1, NE activity was higher and elafin expression was lower, compared with pEGFP-N1-Elafin transfected group (P < 0.05). In pEGFP-N1 transfected cells, LPS suppressed tight junctions protein zonula occludens-1 (ZO-1) production, while in recombinant pEGFP-N1-Elafin cells, LPS did not cause significantly decrease of ZO-1, compared with normal control cells. LPS stimulation also significantly weakened the collagen adhesion capability of pEGFP-N1 transfected cells (P < 0.01), but there was no significant difference in recombinant pEGFP-N1-Elafin cells (P > 0.05). These results suggest that elafin can maintain airway epithelium integrity by protecting airway epithelial cells and enhancing the anti-inflammatory capability of airway.     

Lipopolysaccharide-stimulated activation of murine DC2.4 cells is attenuated by <Emphasis Type="Italic">n</Emphasis>-butylidenephthalide through suppression of the NF-κB pathway

Modulation of dendritic cell (DC) fate and function may be one approach for the treatment of inflammatory and autoimmune diseases. n-Butylidenephthalide (BP), derived from Angelica sinensis, at 40 μg/ml significantly decreased the secretion of interleukin-6 and tumor necrosis factor-α by lipopolysaccharide (LPS)-stimulated activation of cultured murine DC2.4 cells (P < 0.01). LPS-induced major histocompatibility complex class II (P < 0.05), CD86 (P < 0.01) and CD40 (P < 0.01) expression on DC2.4 cells was also inhibited by BP. The endocytic capacity of LPS-stimulated DC2.4 cells was increased by BP (P < 0.01). The antigen-presenting capacity of LPS-stimulated DC2.4 cells was decreased by BP (P < 0.05). Moreover, we confirmed BP attenuates the responses of LPS-stimulated activation of DCs via suppression of NF-κB-dependent pathways.     

Expression of Eph A molecules during swine embryo implantation

Eph–Ephrin system can induce repulsive forces in cell migration and adhesion during embryonic development in various mammals. In this study, the attachment sites of swine endometrium during pregnancy were used and the physiological role of this system in the step of mammalian embryo implantation was estimated to investigate the involvement of the Eph–Ephrin system in swine embryo implantation. Real-time quantitative PCR indicated that mRNA expression of Eph A1 on endometrium increased extremely significantly around the implantation period (P < 0.01), while expression of Eph A2 and A4 decreased significantly during this period (P < 0.05). Immunostaining showed that protein expression of Eph A1, A2 and A4 in the endometrial stroma underlying the luminal epithelium was higher during mid-implantation compared with early or post-implantation. Western blotting examination demonstrated that protein expression of Eph A1, A2 and A4 at the attachment sites of swine endometrium increased from pregnancy day 13 to 18 (P < 0.01), and then decreased from pregnancy day 18 to 24 (P < 0.01). These findings suggest that the Eph–Ephrin A system might play an important role in regulating the swine contact between blastocysts and endometrium during embryo implantation.     

Apolipoprotein A-I inhibits chemotaxis,adhesion, activation of THP-1 cells and improves the plasma HDL inflammatory index

The anti-inflammatory effects of high density lipoprotein (HDL) are well described, however, such effects of Apolipoprotein A-I (ApoA-I) are less studied. Building on our previous study, we further explored the mechanism of anti-inflammatory effects of ApoA-I, and focused especially on the interaction between monocyte and endothelial cells and plasma HDL inflammatory index in LPS-challenged rabbits. Our results show that ApoA-I significantly decreased LPS-induced MCP-1 release from THP-1 cells and ox-LDL-induced THP-1 migration ratio (P < 0.01, respectively). ApoA-I significantly decreased sL-selectin, sICAM-1 and sVCAM-1 release (P < 0.01, P < 0.01, P < 0.05, respectively) from LPS-stimulated THP-1 cells. Furthermore, ApoA-I significantly inhibited LPS-induced CD11b and VCAM-1 expression on THP-1 cells (P < 0.01, P < 0.05, respectively). ApoA-I diminished LPS-induced mCD14 expression (P < 0.01) and NFκB nuclear translocation in THP-1 cells. After single dose treatment of ApoA-I, the value of plasma HDL inflammatory index in LPS-challenged rabbits was improved significantly (P < 0.05). These results suggest that ApoA-I can inhibit chemotaxis, adhesion and activation of human monocytes and improve plasma HDL inflammatory index with presenting beneficial anti-inflammatory effects.     

Intranasally administered Lactobacillus gasseri TMC0356 protects mice from H1N1 influenza virus infection by stimulating respiratory immune responses

We conducted a study to evaluate the possibility that intranasal administration of a new probiotic strain Lactobacillus gasseri TMC0356 (TMC0356) may protect host animals from influenza virus (IFV) infection, which was indicated by enhanced respiratory immune responses in a mouse model. After 3 days of exposure to TMC0356, BALB/c mice were intranasally infected with IFVA/PR/8/34 (H1N1). Lung cells were isolated from the tested mice and evaluated for cytotoxicity against YAC-1 cells. After intranasal treatment with TMC0356, mice showed a lower morbidity and higher survival rate compared to control mice (P < 0.05). The cytotoxicity of lung cells isolated from mice after intranasal treatment against YAC-1 cells was statistically higher than that of lung cells isolated from control mice (P < 0.05). Intranasal administration of TMC0356 significantly increased mRNA expression of interleukin (IL)-1β, tumor necrosis factor, IL-10, and monocyte chemotactic protein-1 (P < 0.01). These results suggest that intranasal administration of TMC0356 may protect the host animal from IFV infection. They also indicate that TMC0356 can enhance respiratory cell-mediated immune responses of host animals characteristically with up-regulated activation of lung natural killer cells. Further studies will evaluate the possible role of the immune stimulatory effects of TMC0356 within the protective effects of this bacterium against IFV, as observed in the present study.     

Effect of silicon on chilling-induced changes of solutes,antioxidants, and membrane stability in seashore paspalum turfgrass

We present a detailed study to investigate if silicon supplementation enhances chilling resistance of seashore paspalum (Paspalum vaginatum Swartz) turf. An enhanced growth status suggests an improved chilling resistance by Si addition, which is coupled with the observation of more Si cells in leaf epidermal cells, as well as a lower LT₅₀ (the low temperature required to cause 50% electrolyte leakage). Chilling stress induces significant adaptive increases of free proline (P < 0.01), all soluble sugar (P < 0.01) and the activity of peroxidase (POD) (P < 0.05), and leads to the decreases of the activities of superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), results in notably higher measurements of malondialdehyde (MDA) (P < 0.05). Silicon addition promoted significant increase of proline and sucrose (P < 0.01), while maintaining significantly higher activities of SOD, POD, CAT, and notably leveling off of MDA (P < 0.05) under chilling stress. These results indicate that silicon enhances the chilling resistance of turfgrass via maintaining a stable membrane and a beneficial cell status readily coping with the chilling-induced oxidative stress.     

Role of iNOS-derived reactive nitrogen species and resultant nitrative stress in leukocytes-induced cardiomyocyte apoptosis after myocardial ischemia/reperfusion

Polymorphonuclear leukocyte (PMN) accumulation/activation has been implicated as a primary mechanism underlying MI/R injury. Recent studies have demonstrated that PMNs express inducible nitric oxide synthase (iNOS) and produce toxic reactive nitrogen species (RNS). However, the role of iNOS-derived reactive nitrogen species and resultant nitrative stress in PMN-induced cardiomyocyte apoptosis after MI/R remains unclear. Male adult rats were subjected to 30 min of myocardial ischemia followed by 5 h of reperfusion. Animals were randomized to receive one of the following treatments: MI/R+vehicle; MI/R+L-arginine; PMN depletion followed by MI/R+vehicle; PMN depletion followed by MI/R+L-arginine; MI/R+1400 W; MI/R+1400 W+L-arginine and MI/R+ FeTMPyP. Ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis were determined. PMN depletion virtually abolished ischemia/reperfusion- induced PMN accumulation, attenuated ischemic/reperfusion-induced and L-arginine-enhanced nitrative stress, and reduced ischemic/reperfusion-induced and L-arginine-enhanced cardiomyocyte apoptosis (P values all <0.01). Pre-treatment with 1400 W, a highly selective iNOS inhibitor, had no effect on PMN accumulation in the ischemic/reperfused tissue. However, this treatment reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis to an extent that is comparable as that seen in PMN depletion group. Treatment with FeTMPyP, a peroxynitrite decomposition catalyst, had no effect on either PMN accumulation or total NO production. However, treatment with this ONOO⁻ decomposition catalyst also reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis (P values all <0.01). These results demonstrated that ischemic/reperfusion stimulated PMN accumulation may result in cardiomyocyte injury by an iNOS-derived nitric oxide initiated and peroxynitrite-mediated mechanism. Therapeutic interventions that block PMN accumulation, inhibit iNOS activity or scavenge peroxynitrite may reduce nitrative stress and attenuate tissue injury. Xiao-Liang Wang and Hui-Rong Liu contributed equally to this study.     

Effect of mechanical deformation on structure and function of polymorphonuclear leukocytes

Kitagawa, Yuko, Stephan F. Van Eeden, Darlene M. Redenbach,Maleki Daya, Blair A. M. Walker, Maria E. Klut, Barry R. Wiggs, andJames C. Hogg. Effect of mechanical deformation on structure andfunction of polymorphonuclear leukocytes. J. Appl.Physiol. 82(5): 1397-1405, 1997.The presentstudies were designed to test the hypothesis that mechanicaldeformation of polymorphonuclear leukocytes (PMN) leads to functionalchanges that might influence their transit in the pulmonarycapillaries. Human leukocytes were passed through 5- or 3-µm-porepolycarbonate filters under controlled conditions. Morphometricanalysis showed that the majority of PMN were deformed and that thisdeformation persisted longer after filtration through 3-µm filtersthan through 5-µm filters (P < 0.05) but did not result in the cytoskeletal polarizationcharacteristic of migrating cells. Flow cytometric studies of thefiltered PMN showed that there was a transient increase in thecytosolic free Ca²⁺ concentrationafter both 3- and 5-µm filtration (P < 0.01) with an increase in F-actin content after 3-µm filtration(P < 0.05). AlthoughL-selectin expression on PMN wasnot changed by either 5- or 3-µm filtration, CD18 and CD11b wereincreased by 3-µm filtration (P < 0.05). Priming of the PMN withN-formyl-methionyl-leucyl-phenylalanine (0.5 nM) before filtration resulted in an increase of CD11b by both 5 (P < 0.05)- and 3-µm(P < 0.01) filtration. Neither 5- nor 3-µm filtration induced hydrogen peroxide production. We conclude that mechanical deformation of PMN, similar to what occurs in thepulmonary microvessels, induces both structural and functional changesin the cells, which might influence their passage through the pulmonarycapillary bed.

     

The protective effect of quercetin on long-term alcohol consumption-induced oxidative stress

Long-term alcohol consumption can cause oxidative stress and cytokines induction, which are associated with free radicals. Quercetin, one of the most widely distributed flavonoids in plants, is a natural antioxidant. We investigated the hypothesis that quercetin could prevent the ethanol-induced oxidative stress and decreases tumor necrosis factor-α (TNF-α) and interferon-γ (INF-γ) as pro-inflammatory cytokines. Twenty-eight rats were randomly divided into control group (C), ethanol treatment group (EtOH) (~1 ml/day, 80%; 2 g/kg body wt), intragastrically (i.g.), quercetin treatment group (Q), (100 mg/kg-body wt per 3 days) i.g. and ethanol plus quercetin treatment group (EtOH + Q) (1 ml/day, 80% of ethanol and 100 mg/kg-body wt of quercetin per 3 days) i.g. for 30 days Plasma thiobarbituric acid reactive substance (TBARS) levels and protein carbonyl content were significantly higher in the EtOH group than the C group (P < 0.01). On the other hand, TBARS level and protein carbonyl content in the EtOH + Q group was decreased significantly by quercetin (P < 0.05, P < 0.01; respectively). While GSH levels in whole blood decreased in EtOH group compared to C group, they increased significantly by quercetin (P < 0.05). Plasma ALT, TNF-α and IFN-γ levels increased significantly in the EtOH group compared to control group (P < 0.05, P < 0.01, P < 0.01, respectively), but they decreased significantly in the EtOH + Q group in comparison with EtOH group (P < 0.05, P < 0.01, P < 0.01, respectively). Our results demonstrate that quercetin treatment may provide a protection as reflected by decreased plasma TBARS, protein carbonyls, TNF-α, INF-γ and ALT levels against ethanol-induced oxidative damage.     

Oxidative stress elevated DNA damage and homocysteine level in normal pregnant women in a segment of Pakistani population

Maternal oxidative stress during pregnancy may impair fetal growth and help in the development of diseases in adulthood. The aim of current study was to assess total oxidation status (TOS), related parameters and their relationship to DNA damage (%) and homocysteine level in normal pregnant women in low-income participants. In a cross-sectional study healthy women were grouped as normal, while age matched nulliparous and singleton pregnancies were included for first, second and third trimester groups. TOS (P < 0.01), melanodialdehyde (MDA) (P < 0.001), aspartate aminotransferase (AST) (P < 0.01), triiodothyronine (T3) (P < 0.01), thyroxine (T4) (P < 0.01), and homocysteine (P < 0.001), in pregnant women were significantly higher as compared to normal healthy women. While serum total proteins (P < 0.01), albumin (P < 0.01) and total antioxidant status (TAS) (P < 0.001) decreased significantly as compared to normal healthy women. Women in third trimester showed a significantly high level of body temperature (P < 0.01), triglyceride (P < 0.01), LDL-cholesterol (P < 0.05), AST (P < 0.01), T3 (P < 0.01), homocysteine (P < 0.001), TOS (P < 0.01) and MDA (P < 0.001) but a lower concentration of serum proteins, albumin and TAS at the end of the pregnancy. Pearson correlation indicated a positive relationship of homocysteine with triglycerides (P < 0.027), TOS (P < 0.01), MDA (P < 0.035) and had a negative relationship with total protein (P < 0.026). DNA damage was strongly related with T3 (P < 0.008), TOS (P < 0.02), MDA (P < 0.037) and MBI (P < 0.048) profiles of pregnant women. These changes were considered normal for pregnant women having optimum blood pressure and normal child birth. Hormonal influences and hemodilution may contribute towards the observed changes in this study.     

Dietary rapamycin supplementation reverses age‐related vascular dysfunction and oxidative stress,while modulating nutrient‐sensing,cell cycle,and senescence pathways

Inhibition of mammalian target of rapamycin, mTOR, extends lifespan and reduces age‐related disease. It is not known what role mTOR plays in the arterial aging phenotype or if mTOR inhibition by dietary rapamycin ameliorates age‐related arterial dysfunction. To explore this, young (3.8 ± 0.6 months) and old (30.3 ± 0.2 months) male B6D2F1 mice were fed a rapamycin supplemented or control diet for 6–8 weeks. Although there were few other notable changes in animal characteristics after rapamycin treatment, we found that glucose tolerance improved in old mice, but was impaired in young mice, after rapamycin supplementation (both P < 0.05). Aging increased mTOR activation in arteries evidenced by elevated S6K phosphorylation (P < 0.01), and this was reversed after rapamycin treatment in old mice (P < 0.05). Aging was also associated with impaired endothelium‐dependent dilation (EDD) in the carotid artery (P < 0.05). Rapamycin improved EDD in old mice (P < 0.05). Superoxide production and NADPH oxidase expression were higher in arteries from old compared to young mice (P < 0.05), and rapamycin normalized these (P < 0.05) to levels not different from young mice. Scavenging superoxide improved carotid artery EDD in untreated (P < 0.05), but not rapamycin‐treated, old mice. While aging increased large artery stiffness evidenced by increased aortic pulse‐wave velocity (PWV) (P < 0.01), rapamycin treatment reduced aortic PWV (P < 0.05) and collagen content (P < 0.05) in old mice. Aortic adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation and expression of the cell cycle‐related proteins PTEN and p27kip were increased with rapamycin treatment in old mice (all P < 0.05). Lastly, aging resulted in augmentation of the arterial senescence marker, p19 (P < 0.05), and this was ameliorated by rapamycin treatment (P < 0.05). These results demonstrate beneficial effects of rapamycin treatment on arterial function in old mice and suggest these improvements are associated with reduced oxidative stress, AMPK activation and increased expression of proteins involved in the control of the cell cycle.     

Mannose receptor-mediated endothelial cell activation contributes to B16 melanoma cell adhesion and metastasis in liver

The role of mannose receptors from hepatic sinusoidal endothelium (HSE) in liver colonization by B16 melanoma (B16M) cells was studied. The expression of high mannose-type oligosaccharides on the surface of B16M cells was enhanced by in vitro treatment with 1-deoximannojirimycin (1-DMM). There was a significant (P < 0.01) enhancement of hepatic metastasis when B16M cells were 1-DMM-treated before being intrasplenically injected into C57BL/6J mice. Intraperitoneal administration of 5 mg/kg recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) inhibited the 1-DMM-induced enhancement of metastasis. Expression of high mannose-type oligosaccharides on the surface of 1-DMM-treated B16M cells and their in vitro adhesion to the HSE was significantly correlated (R = 0.82). The addition of either 100 μg/ml mannan or paraformaldehyde (PFA)-fixed 1-DMM-treated B16M cells to cultured HSE for a period of 12 h significantly (P < 0.01) increased the release of IL-1β from the HSE compared to that liberated by the HSE incubated with either basal medium or PFA-fixed untreated B16M cells. The same HSE treatments also significantly (P < 0.01) increased the degree of adhesion of other B16M cells to HSE, being abrogated by anti-mouse vascular cell adhesion molecule-1 (VCAM-1) antibodies. The conditioned media from HSE cultures, activated by PFA-fixed, 1-DMM-treated B16M cells significantly (P < 0.01) increased B16M cell proliferation when compared to conditioned media from HSE cultures incubated with PFA-fixed, untreated B16M cells. Thus, 1-DMM treatment of B16M cells enhanced the development of hepatic metastasis by IL-1-dependent mechanisms. The mechanism is consistent with in vitro mannose receptor-mediated melanoma cell attachment to the HSE, which subsequently upregulates IL-1β release, VCAM-1-dependent adherence, and melanoma growth factor(s) release by HSE. J. Cell. Physiol. 174:322–330, 1998. © 1998 Wiley-Liss, Inc.     

Evaluation of Markers of Oxidative Stress,Antioxidant Function and Astrocytic Proliferation in the Striatum and Frontal Cortex of Parkinson’s Disease Brains

Dopaminergic neurons die in Parkinson’s disease (PD) due to oxidative stress and mitochondrial dysfunction in the substantia nigra (SN). We evaluated if oxidative stress occurs in other brain regions like the caudate nucleus (CD), putamen (Put) and frontal cortex (FC) in human postmortem PD brains (n = 6). While protein oxidation was elevated only in CD (P < 0.05), lipid peroxidation was increased only in FC (P < 0.05) and protein nitration was unchanged in PD compared to controls. Interestingly, mitochondrial complex I (CI) activity was unaffected in PD compared to controls. There was a 3–5 fold increase in the total glutathione (GSH) levels in the three regions (P < 0.01 in FC and CD; P < 0.05 in Put) but activities of antioxidant enzymes catalase, superoxide dismutase, glutathione reductase and glutathione-s-tranferase were not increased. Total GSH levels were elevated in these areas because of decreased activity of gamma glutamyl transpeptidase (γ-GT) (P < 0.05) activity suggesting a decreased breakdown of GSH. There was an increase in expression of glial fibrillary acidic protein (GFAP) (P < 0.001 in FC; P < 0.05 in CD) and glutathione peroxidase (P < 0.05 in CD and Put) activity due to proliferation of astrocytes. We suggest that increased GSH and astrocytic proliferation protects non-SN brain regions from oxidative and mitochondrial damage in PD.     

The Effect of Selenium Supplementation in the Prevention of DNA Damage in White Blood Cells of Hemodialyzed Patients: A Pilot Study

Patients with chronic kidney disease (CKD) have an increased incidence of cancer. It is well known that long periods of hemodialysis (HD) treatment are linked to DNA damage due to oxidative stress. In this study, we examined the effect of selenium (Se) supplementation to CKD patients on HD on the prevention of oxidative DNA damage in white blood cells. Blood samples were drawn from 42 CKD patients on HD (at the beginning of the study and after 1 and 3 months) and from 30 healthy controls. Twenty-two patients were supplemented with 200 μg Se (as Se-rich yeast) per day and 20 with placebo (baker's yeast) for 3 months. Se concentration in plasma and DNA damage in white blood cells expressed as the tail moment, including single-strand breaks (SSB) and oxidative bases lesion in DNA, using formamidopyrimidine glycosylase (FPG), were measured. Se concentration in patients was significantly lower than in healthy subjects (P < 0.0001) and increased significantly after 3 months of Se supplementation (P < 0.0001). Tail moment (SSB) in patients before the study was three times higher than in healthy subjects (P < 0.01). After 3 months of Se supplementation, it decreased significantly (P < 0.01) and was about 16% lower than in healthy subjects. The oxidative bases lesion in DNA (tail moment, FPG) of HD patients at the beginning of the study was significantly higher (P < 0.01) compared with controls, and 3 months after Se supplementation it was 2.6 times lower than in controls (P < 0.01). No changes in tail moment was observed in the placebo group. In conclusion, our study shows that in CKD patients on HD, DNA damage in white blood cells is higher than in healthy controls, and Se supplementation prevents the damage of DNA.     

Pericardial Fat Volume Correlates With Inflammatory Markers: The Framingham Heart Study

The objective of this study was to determine whether systemic inflammatory and oxidative stress marker concentrations correlate with pericardial and intrathoracic fat volumes. Participants of the Framingham Offspring Study (n = 1,175, 53% women, mean age 59 ± 9 years) had pericardial and intrathoracic fat volumes assessed by multidetector computed tomography (MDCT) scans, and provided fasting blood and urine samples to measure concentrations of 14 inflammatory markers: C‐reactive protein (CRP), interleukin‐6, monocyte chemoattractant protein‐1 (MCP‐1), CD40 ligand, fibrinogen, intracellular adhesion molecule‐1, lipoprotein‐associated phospholipase A₂ activity and mass, myeloperoxidase, osteoprotegerin, P‐selectin, tumor necrosis factor‐α, tumor necrosis factor receptor‐2, and urinary isoprostanes. Multivariable linear regression models were used to determine the association of log‐transformed inflammatory marker concentrations with fat volumes, using fat volume as the dependent variable. Due to smaller sample sizes, models were rerun after adding urinary isoprostanes (n = 961) and tumor necrosis factor‐α (n = 813) to the marker panel. Upon backward elimination, four of the biomarkers correlated positively with each fat depot: CRP (P < 0.0001 for each fat depot), interleukin‐6 (P < 0.05 for each fat depot), MCP‐1 (P < 0.01 for each fat depot), and urinary isoprostanes (P < 0.01 for pericardial fat; P < 0.001 for intrathoracic fat). Even after adjusting for BMI, waist circumference (WC), and abdominal visceral fat, CRP (P = 0.0001) and urinary isoprostanes (P = 0.02) demonstrated significant positive associations with intrathoracic fat, but not with pericardial fat. Multiple markers of inflammation and oxidative stress correlated with pericardial and intrathoracic fat volumes, extending the known association between regional adiposity and inflammation and oxidative stress.     

HSPA12B inhibits lipopolysaccharide‐induced inflammatory response in human umbilical vein endothelial cells

Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 protein family. This study investigated the effects of HSPA12B on lipopolysaccharide (LPS)‐induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms involved. A HUVECs inflammatory model was induced by LPS. Overexpression of HSPA12B in HUVECs was achieved by infection with recombinant adenoviruses encoding green fluorescence protein‐HSPA12B. Knockdown of HSPA12B was achieved by siRNA technique. Twenty four hours after virus infection or siRNA transfection, HUVECs were stimulated with 1 μg/ml LPS for 4 hrs. Endothelial cell permeability ability was determined by transwell permeability assay. The binding rate of human neutrophilic polymorphonuclear leucocytes (PMN) with HUVECs was examined using myeloperoxidase assay. Cell migrating ability was determined by the wound‐healing assay. The mRNA and protein expression levels of interested genes were analyzed by RT‐qPCR and Western blot, respectively. The release of cytokines interleukin‐6 and tumour necrosis factor‐α was measured by ELISA. HSPA12B suppressed LPS‐induced HUVEC permeability and reduced PMN adhesion to HUVECs. HSPA12B also inhibited LPS‐induced up‐regulation of adhesion molecules and inflammatory cytokine expression. By contrast, knockdown of HSPA12B enhanced LPS‐induced increases in the expression of adhesion molecules and inflammatory cytokines. Moreover, HSPA12B activated PI3K/Akt signalling pathway and pharmacological inhibition of this pathway by Wortmannin completely abrogated the protection of HSPA12B against inflammatory response in HUVECs. Our results suggest that HSPA12B attenuates LPS‐induced inflammatory responses in HUVECs via activation of PI3K/Akt signalling pathway.     

The Level of Isoprostanes as a Non-invasive Marker for <Emphasis Type="Italic">in vivo</Emphasis> Lipid Peroxidation in Secondary Progressive Multiple Sclerosis

Oxidative stress leads to lipid peroxidation and may contribute to the pathogenesis of lesions in multiple sclerosis (MS), an autoimmune disease characterized by inflammatory as well as degenerative phenomena. Isoprostanes are prostaglandin-like compounds which are formed by free radical catalysed peroxidation of arachidonic acid esterified in membrane phospholipids. They are a new class of sensitive specific markers for in vivo lipid peroxidation. In this study 26 patients (15 females and 11 males; mean age 48.2 ± 15.2 year; mean disease duration 10.0 ± 6.5 year) with secondary progressive MS (SPMS) and 12 healthy controls were enrolled. In patients with multiple sclerosis the lipid peroxidation as the level of urine isoprostanes and the level of thiobarbituric acid reactive species (TBARS) in plasma were estimated. Moreover, we estimated the total antioxidative status (TAS) in plasma. It was found that the urine isoprostanes level was over 6-fold elevated in patients with SPMS than in control (P < 0.001). In SPMS patients TBARS level was also statistically higher than in controls (P < 0.01). However, we did not observed any difference of TAS level in serum between SPMS patients and controls (P > 0.05). In patients with SPMS the lipid peroxidation and oxidative stress measured as the increased level of isoprostanes was observed. Thus, we suggest that the level of isoprostanes may be used as non-invasive marker for a determination of oxidative stress what in turn, together with clinical symptoms, may determine an specific antioxidative therapy in SPMS patients.     

Fas expression on peripheral blood lymphocytes in systemic lupus erythematosus: relation to the organ damage and lymphocytes apoptosis

Systemic lupus erythematosus (SLE) is a common autoimmune disease with complex etiology. Recently, a possible role of apoptotic cells in its pathogenesis has been suggested. This study is to evaluate the expression of Fas antigen on peripheral blood lymphocytes (PBLs) in SLE, to determine whether membrane Fas (mFas) has a role in the organ damage in SLE and to explore its relationship with the early apoptosis of the PBLs in SLE. Flow cytometry was used to evaluate the expression of mFas on PBLs in 68 Chinese SLE patients and 37 healthy controls. Systemic lupus international collaborative clinics/american colleges of rheumatology damage index (SDI) scores were calculated to further estimate the relationship of the expression of mFas with disease severity. Results showed that mFas expression levels were significantly higher (P < 0.01) among SLE patients than those in healthy controls. Higher (P = 0.01) expressions of mFas were found in patients with SDI scores ≥ as compared to those with SDI scores <3. Patients with neuropsychiatric or renal disease had a higher expression of mFas than those without neuropsychiatric (P = 0.03) or renal disease (P = 0.01). In addition, the expression levels had a positive (r = 0.381, P < 0.01) correlation with the early apoptosis rate of PBLs in SLE patients. Taken together, our study showed that Fas-expressing PBLs were increased in SLE patients, especially in patients with higher SDI score, and the expression levels of mFas were correlated to the organ damage and lymphocytes apoptosis.     

Fibronectin and Focal Adhesion Kinase Small Interfering RNA Modulate Rat Retinal Müller Cells Adhesion and Migration

Retinal Müller cells (RMCs) hypertrophy and proliferation play a crucial role in epiretinal membrane formation. This study was designed to analyze the effects of Fibronectin and specific FAK siRNA in cell adhesion and migration in rat Müller cells. RMCs were cultured and identified by GFAP, Vimentin, and GLAST mAb, respectively. The cells were planted on dishes coated with Fibronectin at 0, 1, 5, 10, 50, and 100 μg/ml. The attachment and migration assay was applied to characterize the RMCs–Fibronectin interactions. Cell lysis and Western blotting were utilized to detect β₁-integrin, FAK, and GLAST protein expression. Then the cells were treated with FAK siRNA, non-targeting siRNA, and control medium. The cell cycle and apoptosis rate was determined by flow cytometry. The attachment, migration, and Western blotting assay were repeated. These data suggested that almost all the cells expressed GFAP, Vimentin, and GLAST, respectively, which ensured most of the harvested cells were RMCs. In attachment assay, the A570 values increased significantly with time (F = 1105.439, P < 0.001) and Fibronectin concentration (F = 424.683, P < 0.001). There were significant difference between each Fibronectin concentration in RMCs migration (F = 34.703, P < 0.000). The expression ratio of FAK, β₁-integrin, and GLAST elevated significantly as Fibronectin concentration increased (F = 54.755, P < 0.000; F = 119.962, P < 0.000; F = 39.287, P < 0.000). The Fibronectin pretreatment was settled on 50 μg/ml for siRNA inhibition assays. The specific FAK siRNA treatment significantly increased G₀/G₁ percentage and apoptosis rate compared with NT siRNA and control group (F = 11.526, P = 0.009; F = 64.772, P < 0.000). The apoptotic rate was significantly suppressed by inhibitors of caspase-8 and 3 (F = 10.500, P = 0.011). The A570 values were significantly suppressed in FAK siRNA groups compared with NT siRNA and control group (F = 154.241, P < 0.000), and the mean migratory cells per view field were significantly decreased (F = 10.906, P = 0.001). FAK and GLAST expression ratio decreased significantly after FAK siRNA treatment (F = 5.315, P = 0.047; F = 5.985, P = 0.042). Take together, FAK is involved in β₁-integrin mediated adhesive signaling and play a critical role in regulating Müller cell adhesion, migration, and so far as to glutamate transportation functions.     

Chromium Supplementation Can Alleviate the Negative Effects of Heat Stress on Growth Performance,Carcass Traits,and Meat Lipid Oxidation of Broiler Chicks without Any Adverse Impacts on Blood Constituents

This study was conducted to determine the effects of dietary supplementation with Cr nicotinate and Cr chloride and their optimum inclusion rate on performance, carcass traits, meat oxidative stability, serum metabolites, hematological parameters, and liver chromium concentration in heat-stressed broilers. A total number of 420, 1-day-old male broiler chicks were randomly assigned to seven treatments with four replicates of 15 chicks. The dietary treatments consisted of the basal diet supplemented with 0 (control), 500, 1,000, and 1,500 μg/kg Cr in the form of Cr nicotinate and Cr chloride. Chicks were raised for 6 weeks in heat stress condition (33 ± 2°C). Supplements of organic and inorganic Cr particularly at 1,500 μg/kg incorporation increased feed consumption (P < 0.05) and body mass gain of broilers (P < 0.01). Cr supplementation increased carcass yield and decreased abdominal fat (P < 0.01). Supplementation of 1,500 μg/kg Cr nicotinate (P < 0.05) enhanced liver Cr concentration. Storage time increased lipid oxidation of meat (P < 0.01). Cr decreased lipid oxidation of breast and thigh muscles over 2 (P < 0.01) or 6 (P < 0.05) days of storage time. Birds fed 1,500 μg/kg Cr nicotinate, had lower concentration of serum glucose and triglyceride at 21 days (P < 0.05). Hematological parameters tested at 21 and 42 days, were not influenced. The results suggested that dietary Cr supplementation regardless of its source have a positive effect on productive, and carcass traits, also enhances oxidative stability of refrigerated meat in broilers reared under heat stress conditions.