Objective To investigate the influence of panton-valentine leucocidin (PVL) on expression of Toll like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signals and IL-8,IL-6 in THP-1 macrophages,and to study the mechanism of PVL-related lung tissue damage.Methods THP-1 cells were cultured in the presence of 100 nmol/L phorbol-12-myristate 3-acetate (PMA) for 48 h to induce monocytemacrophage differentiation.rPVL-F and rPVL-S were induced and expressed from the recombinant plasmid,respectively purified with chromatographic column. After that,THP-1 macrophages were incubated with rPVL,and then ELISA was performed to test expression of IL-8 and L-6 in supernatants fluid; RT-PCR was performed to detect expression of IL-8,L-6 and TLR4 ; NF-κB was analyzed by Western blot and immunohistochemistry method.Results PVL was able to induce expression of IL-8 and IL-6 in THP-1 macrophages in time-and concentration-dependent manners.PVL could also significantly promote the activation of TLR4/NF-κB signals.Conclusion PVL can activate the expression of TLR4/NF-κB signals,and increased the high expression of inflammatory cytokines.Maybe it's the mechanism of action of PVL exerts the function of lung tissue damage.

Microglia are the principal immune effectors in brain and participate in a series ofneurodegenerative diseases. The microglial shapes are highly plastic. The morphology is closely related with their activation status and biological functions. Cerebral ischemia could induce microglial activation, and microglial activation is subjected to precise regulation. Microglia could play either protective or neurotoxic roles in cerebral ischemia. Therefore, regulating the expression of receptors or protein molecules on microglia, inhibiting the excessive activation of microglia and production of pro-inflammatory factors, promoting the release of neuroprotective substances might be beneficial to the treatment of cerebral ischemia. The study about relationship between microglia and cerebral ischemia will shed a light on the treatment of cerebral ischemia. This paper is a review of microglial activation and regulation during cerebral ischemia as well as related therapeutic methods.

Background Sphingosine-l-phospate (S1P) is a bioactive lipid and important messenger molecule in cells.It participates in the regulation of many biological processes,such as cell proliferation,migration,survival,differentiation,apoptosis,etc.Hypoxia is a trigger factor of choriod neovascularization (CNV) and pathological basis of many diseases,and retinal pigment epithelial (RPE) cells are involved in formation of CNV.However,the effects of S1P on proliferation and apoptosis of RPE cells are below understood.Objective This study was to investigate the influence of S1P on proliferation and apoptosis of human RPE cells under hypoxic conditions.Methods Human RPE cells line-D407 cells were cultured and passaged and generation 3-5 cells were used and divided into 6 groups.The cells were regularly cultured in the blank control group using DMEM containing 10％ fetal bovine serum.CoCl2(200.00 μmol/L) was added into the colture medium for 2 hours in the hypooxic group.S1P of different concentrations (0.01,0.10,1.00,10.00 μmol/L) were added in culture medium 2 hours after the affection of 200.00 μmol/L CoCl2.The proliferative values of the cells were detected using WST-1 method as the absorbance (A value) and the proliferative rate of different groups were calculated.The apoptosis of the cells was assayed by Hoechst staining.The results were compared among different groups.Results Cultured cells showed the round-like in shape with clear nuclei and pigment.The proliferative values (A value) was 0.91 ±0.08,0.37±0.09,0.46±0.08,0.52±0.09,0.61 ±0.06,0.70±0.10 in the blank control group,hypoxic group and 0.01,0.10,1.00,10.00 μmol/L S1P groups,respectively,with a significant difference among the groups (F=21.104,P=0.000),and A values in various S1P groups were higher than those in the hypoxiac group (all at P＜0.05).The proliferative rate was gradually raised with the increase of dose of S1P.Hoechst staining exhibited a few apoptosis cells in the blank control group,but in the hypoxic group,a lots of apoptosis cells were seen with the light-blue nuclei and condensable chromatin.However,the number of apoptosis cells was significantly decreased in various concentrations of S 1 P groups.The apoptosis rates were (1.21 ±0.08) ％,(8.99 ±0.09) ％,(6.60 ±0.08) ％,(5.95 ±0.09) ％,(4.81 ± 0.06)％ and (3.96±0.10)％ in the blank control group,hypoxic group and the 0.01,0.10,1.00,10.00 μmol/L S1P groups,respectively,with a significant difference among the groups (F =25.070,P =0.000).Compared with the hypoxia group,the cellular apoptosis rates of various S1P groups were lower (all at P＜0.05).Conclusions Under the hypoxia condition,S1P can promote the proliferation of human RPE cells and inhibit apoptosis.

ObjectiveTo explore the role of polymorpbonuclear leukocyte (PMN) in PantonValentine leucocidin (PVL)-induccd acute lung inflammation and injury. Methods Fifteen New Zealand rabbits were divided into 3 groups with five rabbits in each group.The controls were treated with pbosphate buffer solution (PBS),the rabbits with normal granulocyte in rPVL group were treated with endotracheal instillation of rPVL,the granulocytopenia rabbits in vincristine (VCR) +rPVL group were firstly treated with VCR,thcn with endotracheal instillation of rPVL.Nine hours after injection,the peripheral blood and bronchoalveolar lavage fluid (BALF) were collected for counting PMN.The lactate dehydrogenase (LDH) activity in BALF,lung permeability index (LPI),PMN apoptosis and necrosis and the release of reactive oxygen species (ROS)in BALF were measured.After the rabbits sacrificed,the lung tissue samples were collcctcd for dctcrmining wet/dry (W/D) ratio and histopathological examination.The comparison among groups was done by t test.ResultsThe PMN count in the peripheral blood was (2.69=0.34) × 10 mL in rPVL group,which was significantly lower than control group [(3.63 ± 0.38) × 105/mL] (t =4.12,P＜0.05).The PMN counts in BALF in control group,rPVL group and VCR+rPVL group were (0.57±0.01 ) ×106/mL,(3.01±0.02) × 106/mL and (0.10±0.02) × 106/mL,respectively; that in rPVL group was significantly higher than those in control group (t=254.39,P＜0.05).The LDH activity,LPI and W/D ratio in rPVL group were all significantly higher than control group,while those in VCR+rPVL group were not significantly different from control group.The PMN apoptosis rate and necrosis rate in VCR+rPVL groupwere (1.17±0.24)％ and (1.13±0.17)％,respectively.The releases of ROS (meanfluorescence intensity) in rPVL group,control group and VCR+rPVL group were 1.56±0.39,0.41±0.03 and 0.39±0.02,respectively,and that in rPVL group was significantly higher (t=6.58,P＜0.05).Histopathological examination of the lung showed the diffuse infiltration of inflammatory cells,hemorrhage and edema in rPVL group,wbile there was only thimbleful infiltration of inflammatory cells observed in surrounding bronchia and alveolar septun in VCR-rPVL group.ConclusionsrPVL can induce lung inflammation and injury in rabbits with normal granulocyte,but not in neutropenic rabbirs.Lung inflammation and injury may be the result of recruitment,aggregation and subsequent lysis and/or activation of PMN,which can damage the lung by releasing the contents of cytotoxic granules and/or reactive oxygen metabolites.

ObjectiveTo explore the role of NF-κB signaling pathway protein and cytokines in Panton-Valentine leukocidin (PVL)-induced acute lung inflammation and injury.MethodsThirty rabbits were distributed randomly into two groups,each group had fifteen rabbits.Group rPVL were directly treated with endotracheal instillation of rPVL,normal control were treated with PBS.Then five rabbits chosen at random from each group were killed at 3,6,or 9 h postinfection.The lung was removed from the rabbits to determine histopathology studies.ELISA was performed to evaluate levels of IL-6,IL-8,IL-10 and TNF-α.NF-κB p65 protein of the lung tissue was assessed by immunohistochemistry method.ResultsIn group rPVL histopathology study showed symptoms of severe illness:diffuse infiltration of inflammatory cells,hemorrhage,edema and other manifestations of lung injury.Levels of IL-6,IL-8 and TNF-α were increased gradually,and the level of IL-10 was increased at 9 h postinfection.The expression of NF-κB p65 protein was increased gradually with the infection time.ConclusionNF-κB activation and cytokines release play an important role in PVL-related lung injury.It may be an important path to down regulate the counts of NF-κB activation.

Background Several types of cells participate in the formation of proliferative membrane in proliferative retinopathy (PVR),and the proliferation,migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells play an important role.Many studies have confirmed high blood glucose is the basic pathogenesis of diabetic retinopathy (DR).However,whether EMT could be induced in RPE cells under the high glucose condition has not been reported.Objective This study was to investigate the effects of high glucose on the migration and EMT of RPE cells in high glucose culture model in vitro.Methods Human RPE cell line D407 were cultured and passaged in DMEM/F12 medium with 10％ fetal bovine serum,and 6-8 generations of cells were used in experiment.The cells were divided into 3 groups based on different glucose concentrations in medium.The glucose at the final concentration 5.5 mmol/L or 60.0 mmol/L was respectively used in the normal control group or high glucose group,and the DMEM with 5.5 mmol/L glucose and mannitol was used in the hypertonic control group.The migration rate of the cells were detected 0,24,48 and 72 hours after scratching by wound-scratch test.Real-time PCR was used to detect the relative expressions of zonula occludens-1 (ZO-1) and α-smooth muscle actin (α-SMA) in the cells.Results Cultured cells showed a polygon shape with the clear nucleolus and dense arrangement in the normal control group and the hypertonic control group,but the cells were larger and elongated with the lapse of culture time with the indistinct structure and loose arrangement.At 48 hours after scratching,migrating cells were seen in the scratching area,and the scratching area disappeared at 72 hours after scratching in the high glucose group,but the scratching area still was existed in the normal control group or hypertonic control group.The migrating rate of the cells was higher in the high glucose group than that in the normal control group or hypertonic control group,showing total differences among 3 groups and various time points (Fgroup =328.600,P =0.000 ; Ftime =773.270,P=0.000).Compared with the normal control group,the expression level of ZO-1 mRNA was significantly lower,and α-SMA mRNA level was higher 48 hours and 72 hours in the high glucose group than those in the normal control group (all at P＜0.05).Conclusions High glucose induce the migration and EMT of RPE cells in vitro,which may be associated with the pathogenesis of proliferative diabetic retinopathy.

Objective To investigate the effects of human IL-18 gene combined with diterpenoid alkaloids in inhibiting the proliferation and inducing the apoptosis of tongue squamous carcinoma cells Tscca.Methods We constructed recombinant plasmid pEGFPN3-IL-18 and tranfected it into tongue squamous carcinoma cells Tscca.The transduction efficiency of the target cells was detected by fluorescent microscopy,cytotoxic effect of IL-18 gene with diterpenoid alkaloids on Tscca was detected by MTT assay,and apoptosis was detected by flow cytometry. Western blot was employed to examine the expression level of cellular signal-regulated kinase Akt/p-Akt.Results The tongue squamous cells Tscca which transfected pEGFPN3-IL-18 had significantly increased apoptosis compared with non-transfected cells (P<0 .05 ).Tongue carcinoma squamous cells cultured with diterpenoid alkaloids at the concentrations of 0 .2 ,0 .4 and 0 .6 mg/mL had significantly increased apoptosis in a dose-dependent manner (P<0.05).Human IL-18 gene combined with diterpenoid alkaloids for 48 hours inhibited significantly Tscca in a concentration-dependent manner compared with diterpenoid alkaloids alone (P<0 .05 ).The two in combination could also decrease the protein level of p-Akt dose-dependently.Conclusion The combination of pEGFPN3-IL-18 and diterpenoid alkaloids has a synergistic effect in inhibiting the growth of tongue squamous carcinoma cells Tscca.

Objective To investigate effect of PDTC on the NF-κB activation and the expression of inflammatory cytokines in THP-1 macrophages induced by rPVL.Methods The study was divided into three groups:PBS-treated control group,rPVL-treated group and PDTC group which was given 100 μmol/L PDTC at 60 min before rPVL exposure.Immunohistochemistry method was used to test the translocation of NF-κB protein; the expression of NF-κB and IκB protein was analyzed by Western blot; RT-PCR and ELISA was performed to test expression of IL-8 and L-6 in THP-1 macrophages.Results Compared with rPVL-treated group,the activation of NF-κB and the expression of IL-8 and L-6 in PDTC group was significantly decreased.The protein secretions of IL-8 and IL-6 were reduced to 6.78 ng/ml,3.88 ng/ml,receptively(P ＜0.05).Conclusion The inhibitor of NF-κB,PDTC,could significantly decrease the secretion of pro-inflammatory in THP-1 macrophages by rPVL,and it suggested that PDTC played an important role in protecting tissues from damage induced by rPVL.

Objective To examine the expression of stromal cell-derived factor-1 (SDF-1) in a rat model of retinal ischemia-reperfusion injury (RIRI),and investigate the protective effect of 17β-Estradiol (E2) on RIRI and explore the mechanism.Methods The RIRI model was established in Sprague-Dawley rats by increasing the intraocular pressure.Relative expression levels of SDF-1 mRNA and protein in the retina at 6 hours,12 hours and 24 hours following reperfusion was determined by RT-PCR and Western blot,respectively.E2 was administered to investigate the effects of estrogen on SDF-1 expression,and the estrogen receptor antagonist ICI 182-780 was administered to investigate the effect of estrogen receptor on the expression of SDF-1.Results SDF-1 expression in RIRI 6 hours group,12 hours group and 24 hours group was increased compared with normal control group (all P ＜ 0.05),with maximum expression at RIRI 12 hours group.As expected,pretreatment of RIRI rats with E2 had a protection on RIRI retina;SDF-1 expression was increased in RIRI + E2 group compared with IR control group and RIRI + vehicle group (all P ＜ 0.05).RIRI + E2 + ICI 182-780 group could decrease SDF-1 expression compared with RIRI + E2 group(all P ＜ 0.05).Conclusion E2 offers protection against RIRI by inducing an up-regulation in SDF-1 expression through activation of the estrogen receptor.

Objective To determine the potential correlation of 5-HTT gene polymorphisms (5-HTTLPR and STin2) with clinical manifestations in depression.Methods A total of 401 depressed patients,all of Chinese Han region,were collected and genotyped by polymerase chain reactions (PCR).All patients were evaluated using a 17-item Hamilton depression rating scale (HAMD-17) and Hamilton anxiety rating scale (HAMA),and then associated analysis was applied.Results (1) The age of onset in patients with L/S genotype of 5-HTLPR polymorphism were much younger than that of patients with L/L and S/S genotype (F=3.281,P=0.039).Besides,there was also a significant difference of HAMA1 (anxious mood) scores among patients with different genotypes for 5-HTTLPR polymorphism,where the scores of those with L/S genotype were the highest (2.34±0.80,P=0.010).(2) The scores of HAMD10 (mental anxiety),HAMA1 (anxious mood),HAMA3 (fear) and mental anxiety factor were higher in patients with 12/10 genotype than patients with 12/12 and 10/10 genotype for STin2 polymorphism (2.40±0.83,2.38±0.90,1.42± 1.04,14.60±4.26 respectively;P value:0.014,0.044,0.03 and 0.006 respectively).The scores of HAMD10(mental anxiety) and mental anxiety factor were (2.11±0.77),(12.96±3.78) in the 12/12 genotype patients,and significantly lower than that in the 12/10 genotype patients (adjustedPvalue:0.018,0.006).Conclusions A positive association of the 5-HTT polymorphisms with anxious symptoms in depressed patients is revealed.These findings might provide some evidences for the clinical phenotype and optimization of depression treatment.