Effect of S-nitrosylation induced by IL-1β and IFN-γ on DNA binding activity of cAMP response element binding protein in fibroblast-like synoviocytes

AIM: To investigate the effect of S-nitrosylation induced by recombinant interleukin-1β (rIL-1β) and recombinant interferon-γ (rIFN- γ) on DNA binding activity of cAMP response element binding protein (CREB) in fibroblast-like synoviocytes (FLSs). METHODS: (1)FLSs were incubated with rIL-1β and rIFN-γ in the presence or absence of inducible nitric oxide synthase inhibitor aminoguanidine (AG) for 12 h. The supernatant of the cell culture was collected to determine the contents of nitric oxide (NO). The total proteins prepared from each group ［only the total proteins prepared from rIL-1β+rIFN-γ group was reacted with dithiothreitol (DTT) for 15 min in vitro］ were subjected to the biotin switch assay, and the level of S-nitrosylation was determined by Western blotting. (2)FLSs were incubated with rIL-1β, rIL-1β+ rIFN-γ and AG respectively for 12 h. The nuclear extracts from each group were prepared. The nuclear extracts from each group were subjected to electrophoresis mobility shift assay to analyze the DNA binding activity of CREB (only the nuclear extracts from rIL-1β +rIFN-γ group was reacted with DTT for 15 min in vitro before assaying). RESULTS: rIL-1β plus rIFN-γ increased the production of NO and the level of S-nitrosylation, which was inhibited by AG. Administration of DTT in the total proteins reversed the induction of S-nitrosylation by rIL-1β and rIFN-γ. Co-incubation with rIL-1β and rIFN-γ inhibited the CREB activity induced by rIL-1β alone, which was reversed by AG. Administration of DTT in the nuclear extracts reversed the effect of co-incubation of the cytokines. CONCLUSION: Co-incubation with rIL-1β and rIFN-γ may increase the level of S-nitrosylation through inducing the production of endogenous NO, leading to reversible thiols modification of CREB and inhibit the DNA binding activity of CREB in FLSs.     

Effect of high mobility group box protein 1 on expression of STAT/SOCS/cyclin D1 in rat class fibroblast-like synovial cells

AIM: To investigate the regulatory effect of high mobility group box protein 1 (HMGB1) on STAT/SOCS signal transduction in RSC-364 synoviocytes. METHODS: RSC-364 cells induced by 10 μg/L human recombinant HMGB1 were collected at 6 h, 12 h or 24 h after treatment, respectively. The cells without any treatment were also collected at same time points for normal control. The mRNA expression of STAT 1/3 was analyzed by RT-PCR. The phospho-STAT 1/3 (p-STAT 1/3) and STAT 1/3 proteins were detected by Western blotting. The flow cytometric analysis (FCM) was applied to detect the protein expression of p-STAT 1/3, SOCS 1/3 and cyclin D1. RESULTS: HMGB1 at concentration of 10 μg/L significantly up-regulated the mRNA and protein levels of STAT1 expression, as well as cyclin D1 protein, and markedly increased the protein expression of p-STAT1 (P<0.01). Compared to control group, no significant difference of STAT3 and p-STAT3 expression was observed. HMGB1 elevated the protein expression of SOCS1, while the protein level of SOCS3 was only up-regulated at 6 h. CONCLUSION: HMGB1 may activate the process of STAT1 signal transduction and up-regulate the gene expression of cyclin D1, suggesting the role of HMGB1 in promoting the proliferation of synoviocytes.     

农杆菌介导霞多丽葡萄胚性细胞系遗传转化条件的优化

为建立葡萄(Vitis vinifera)遗传转化技术体系,以霞多丽葡萄(Chardonnay)胚性细胞系为靶组织,采用GUS检测法,对影响农杆菌介导葡萄遗传转化效率的主要因素进行了研究。结果表明,超声波处理时间长短对转化效率有较大影响,在所试的0.5、1、5、10 min 4种不同时间的超声波处理中,以5 min时转化效率较好,平均达到9.11个蓝色斑点;AS浓度对转化效率有明显差异,当浓度为50μmol/L时,平均蓝色斑点数为8.89个,100μmol/L时,达到12.44个;DTT质量浓度对转化效率也有较大影响,在所试的3种质量浓度(1,2,3 mg/L)中,以3 mg/L为最佳,有16.67个蓝色斑点。通过GUS瞬时表达检测,确立了农杆菌介导葡萄胚性细胞系遗传转化的几个最适影响因素,从而为葡萄遗传转化技术体系的建立奠定了基础。     

Effects of rosiglitazone on the oxidative stress injury in endothelial outgrowth cells caused by asymmetric dimethylarginine

AIM: To observe the effects of rosiglitazone on the oxidative stress injury in endothelial outgrowth cells (EOCs), which caused by asymmetric dimethylarginine(ADMA). METHODS: The mononuclear cells were harvested from umbilical cord blood by density gradient centrifugation, and induced into EOCs and expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The second generation of EOCs was treated with 10 μmol/L ADMA and different concentrations of rosiglitazone (0, 1, 5, 10 μmol/L) for 72 h. Then the cells were harvested and the apoptosis rate, reproductive activity and vasculogenesis activity were measured by flow cytometry, MTT assay and Matrix gel vasculogenisis assay, respectively. The endothelial nitric oxide synthase gene expression was assayed by RT-PCR. RESULTS: EOCs possessed many endothelial characteristics. Immunostaining showed that the surface antigen factor VIII, CD34 and Flk-1 were positive. The fluorescent staining experiment revealed that EOCs both bound to FITC-UEA-1 and up-took DiI-ac-LDL. Incubation of EOCs with ADMA increased the apoptosis rate and inhibited the reproductive activity and vasculogenesis activity in the cells. Rosiglitazone at concentrations of 1 and 5 μmol/L counteracted such inhibition and stimulated reproductive activity in EOCs (P<0.01), while such protective effects were attenuated or abolished at concentration of 10 μmol/L. In addition, the vasculogenesis activity was inhibited by 10 μmol/L rosiglitazone cooperated with ADMA. RT-PCR assay revealed that eNOS gene expression in 5 μmol/L group was up-regulated and that in 10 μmol/L group was down-regulated. CONCLUSION: Rosiglitazone at lower concentration (<10 μmol/L) protects EOCs from the oxidative stress injury caused by ADMA, and stimulates reproductive activity of EOCs. Such protective effects are partially achieved through the up-regulation of eNOS gene expression.     

Effect of curcumin on the proliferation and apoptosis in LNCap cells

AIM:To observe the effect of curcumin on the proliferation and apoptosis of prostate cancer cell line LNCap. METHODS:LNCap cells were treated with different doses (10 μmol/L, 20 μmol/L, 30 μmol/L, 40 μmol/L) of curcumin and its effects were analyzed in cell growth and apoptosis by microscope, MTT colorimetric assay and flow cytometry. The expression of prostate specific antigen (PSA) was measured by AXSYMTM system-chemical luciferase methods and expression of androgen receptor (AR) was detected by Western blotting. RESULTS:The results showed that curcumin inhibited the proliferation of LNCap cells. The cell growth was inhibited by curcumin in a dose dependent manner and the optimal dose and time was 40 μmol/L, 24 h. Curcumin induced apoptosis in LNCap cells, the most dramatic dose was 40 μmol/L curcumin, at this dose the apoptosis rate was 9.23%. Curcumin inhibited the expression of PSA in LNCap cells and the most dramatic dose and time was 40 μmol/L, 24 h. The PSA in this group was 20% of the control group. Curcumin inhibited the expression of AR on prostate cancer cells. CONCLUSION:Curcumin decreases proliferation and induces apoptosis in LNCap cells in a dose-dependent manner. Curcumin also inhibites the expression of PSA and AR on LNCap cells.     

Effects of leptin on hypoxia-reoxygenation induced apoptosis in human L02 cells and expression of Fas and FasL

AIM: To observe the effect of leptin (LEP) on hypoxia-reoxygenation induced apoptosis in L02 cells.METHODS: In the experiment, L02 cell injury was induced by hypoxic air (95%N2 and 5% CO2). The cultured L02 cells were divided into hypoxic 12 h group (IR group) alone, normal control group and the hypoxic plus leptin (100 μg/L, 200μg/L, 400 μg/L, 800 μg/L and 1 600 μg/L) treatment groups in vitro. Flow cytometry, terminal dUTP nick-end labeling (TUNEL) assay and fluorescent quantitative PCR were used to measure the changes of apoptosis in L02 cells and expression of Fas/FasL mRNA.RESULTS: (1) The percentage of L02 cells apoptosis and positive TUNEL cells significantly increased in IR group compared to control group (P<0.01). When L02 cells were treated with LEP, the percentage of cell apoptosis and positive TUNEL cells were decreased compared to IR group. (2) Compared to control group, the Fas/FasL mRNA expression significantly increased in IR group (P<0.01). When L02 cells were treated with LEP, the Fas/FasL mRNA expression decreased compared to IR group, the effect of LEP at concentration of 400 μg/L was obviously (P<0.05). CONCLUSION: LEP decreases the apoptosis of hypoxic-reoxygenation L02 cells by down-regulation of Fas/FasL mRNA expression in L02 cells.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbonate on the proliferation and apoptosis in K562 cells

AIM: To investigate the effect of pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB on the proliferation and apoptosis of K562 cells and to explore the anti-tumor mechanism of PDTC.METHODS: Trans AMTM NF-κB p65 kit was used to detect the activity of p65 in K562 cells treated by PDTC. The effect of PDTC on the proliferation of K562 cells was measured by WST-1 method. DNA damage was detected by single cell gel electrophoresis (comet assay). The procaspase-3 and activated protein level of caspase-3 were detected by Western blotting.RESULTS: The activity of p65 in K562 cells was inhibited after treated by PDTC (P<0.01). Simultaneously the cell proliferation was significantly inhibited in a dose-and time-dependent manner (P<0.01). The degree of DNA damage in K562 cells treated with PDTC at concentrations of 25 μmol/L, 50 μmol/L or 100 μmol/L was more severe than that in control. The rates of comet cells in the PDTC-treated groups (43.50％, 84.00％, 95.63％) were significantly higher than those in control (9.75％, P<0.01), and it was also dose-dependent. The expression of procaspase-3 and activated caspase-3 protein were detected in the cytoplasm of the K562 cells treated by PDTC by Western blotting.CONCLUSION: NF-κB plays an important role in regulating cell proliferation and apoptosis in K562 cells. PDTC inhibits NF-κB activity and elevates the expression of caspase-3, which is related to increase in cell apoptosis.     

Effect of oxidative damage on expression of endoplasmic reticulum stress-related proteins in human umbilical vein endothelial cells

AIM: To investigate the effects of oxidative damage on the expression of endoplasmic reticulum stress-related proteins in human umbilical vein endothelial cells (HUVECs) exposed to tert-butylhydroperoxide (t-BHP). METHODS: HUVECs were cultured in DMEM medium with 10％FBS and induced by t-BHP at concentrations ranging from 25 to 400 μmol/L for 1-24 h. MTT assay was used to measure the cell viability after treatment with t-BHP. The apoptotic rate was evaluated by fluorescence microscopic analysis with Hoechst/PI staining. The endoplasmic reticulum stress-related proteins were detected by Western blotting.RESULTS: Under the conditions of exposure to t-BHP at concentrations ranging from 25 to 400 μmol/L for l-24 h, the viabilities of HUVECs were gradually reduced in dose-dependent and time-dependent manners. The results of Hoechst/PI staining showed that the ratio of apoptotic cells was increased gradually under the conditions of exposure to t-BHP at concentrations of >25 μmol/L for 8 h. The upregulations of several ER stress-related proteins, including inositol requiring enzyme 1α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, and phospho-c-Jun NH2-terminal kinase (p-JNK), a downstream effector protein of ER stress, and subsequently, caspase-3 were observed by Western blotting. CONCLUSION: The results suggest that oxidative stress mediates apoptosis in HUVECs, which might be correlated to the expression of endoplasmic reticulum stress-related proteins.     

Role of ERK1/2-STAT3 pathway in adaptive cytoprotection induced by H2O2 preconditioning

AIM:To explore the role of extracellular signal-regulated kinases ERK1/2-STAT3 pathway in adaptive cytoprotection induced by H₂O₂ preconditioning in PC12 cells. METHODS:In PC12 cells, the experimental model of cytoprotection by H₂O₂ preconditioning against oxidative stress-induced injury was set up. The morphological changes in the apoptotic cells were observed by using of chromatin dye Hoechst 33258. The percent of apoptotic cells was determined by flow cytometry (FCM) with propidium iodide staining. The levels of p-ERK1/2 and p-STAT3 expression were detected by Western blotting assay. RESULTS:Preconditioning with H₂O₂ at concentration of 100 μmol/L for 90 min obviously inhibited apoptosis induced by 300 μmol/L H₂O₂, and both ERK1/2 and STAT3 were activated. UO126 (10 μmol/L, an inhibitor of ERK1/2) or AG-490 (10μmol/L, an inhibitor of JAK2) significantly blocked the cytoprotection effect of H₂O₂ preconditioning. Moreover, UO126 (10 μmol/L) also markedly inhibited the up-regulation of p-STAT3 expression by H₂O₂ preconditioning. CONCLUSION:H₂O₂ preconditioning activates ERK1/2-STAT3 signal pathway, which may be one of the mechanisms underlying H₂O₂ preconditioning-induced cytoprotection.     

Inhibitory effect of geinistein on apoptosis in hUVECs induced by MCP-1

AIM: To study the inhibitory effect of genistein on apoptosis in human umbilical vein endothelial cells (hUVECs) induced by monocyte chemotactic protein-1 (MCP-1). METHODS: The hUVECs were cultured in vitro and identified. Growth-arrested hUVECs were stimulated with genistein at different concentrations (0.1 μmol, 1.0 μmol, 10 μmol, 100 μmol) and co-treated with MCP-1 (10 μg/L). The survival rates of hUVECs were detected by MTT assay. The cell cycle and DNA content were detected by flow cytometry. To explore the possible mechanism of the genistein interventions, the expressions of Bcl-2, Fas and Bax proteins were detected by flow cytometry and Western blotting.RESULTS: Genistein increased the survival rate and the level of Bcl-2, inhibited Fas and Bax, decreased the ratios of apoptosis compared with MCP-1-induced hUVECs apoptotic group in a dose-dependent-manner. CONCLUSION: Genistein inhibits the apoptosis induced by MCP-1 and the inhibitory effect was relative to the dose of genistein. Its mechanism might be involved in the down-regulation of Fas and Bax expressions and the up-regulation of Bcl-2.     

Effect of berberine on endoplasmic reticulum stress-autophagy pathway in human ovarian cancer SKOV3 cells

AIM: To investigate the regulatory effect of berberine on the endoplasmic reticulum stress-auto-phagy pathway in human ovarian cancer SKOV3 cells. METHODS: Human ovarian cancer SKOV3 cells were cultured in vitro, and berberine at doses of 12.5, 25, 50, 100, 200 and 400 μmol/L were added. After exposure for 12 h, 24 h and 48 h, the viability of the SKOV3 cells was measured by MTT assay. The cells were divided into control group, berberine (50 μmol/L) group, berberine (100 μmol/L) group, and berberine (200 μmol/L) group. After treatment with berberine for 24 h, the effects of berberine on the morphological changes of SKOV3 cells were observed under inverted phase-contrast microscope. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) and ubiquitin-binding protein p62 was observed by indirect immunofluorescence method under laser confocal microscope. The protein expression of beclin-1,LC3,p62, CCAAT/lenhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) was determined by Western blot. RESULTS: Berberine at 12.5, 25, 50, 100, 200 and 400 μmol/L significantly decreased the viability of SKOV3 cells at 12 h, 24 h and 48 h, and the IC₅₀ values of 12 h, 24 h and 48 h were (764.7±0.3) μmol/L, (231.6±0.1) μmol/L and (96.2±0.1) μmol/L, respectively. Laser confocal microscopy showed that the LC3 and p62 proteins were scattered and the fluorescence intensity was increased, while the point-like aggregation was also observed. Berberine at 200 μmol/L obviously enhanced the co-localization of LC3 and p62 proteins. Compared with control group, the expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP, and autophagy-related proteins beclin-1, LC3 and p62 in berberine (200 μmol/L) group was increased significantly (P<0.05). CONCLUSION: Berberine may promote endoplasmic reticulum stress in SKOV3 cells by regulating autophagy.     

Akt and ERK1/2 are involved in brain derived neurotrophic factor-induced angiogenesis of endothelial cells

AIM: To investigate the role of PI3K/Akt and MEK1/ERK pathway in the brain derived neurotrophic factor(BDNF)-induced angiogenesis in vitro and to explore the further molecular mechanisms. METHODS: The phosphorylations of Akt and ERK1/2 were detected in human umbilical vein endothelial cells(HUVECs) by Western blotting. The angiogenic activity in vitro was evaluated by transwell migration assay and tubule formation assay. Cell proliferation was determined by crystal violet staining. Cell apoptosis was analysed by FITC-Annexin-V/PI double staining and flow cytometry. RESULTS: BDNF activated the PI3K/Akt and MEK1/ERK pathway in a time-dependent manner. Ly294002 and PD98059 blocked the activation of Akt and ERK1/2 in response to BDNF. BDNF at concentration of 100 μg/L significantly increased HUVECs tube formation, migration and proliferation in vitro to a degree similar to those induced by 25 μg/L VEGF. Furthermore, tube formation and migration of HUVECs toward BDNF were significantly inhibited by treatment with 20 μmol/L Ly294002 and 20 μmol/L PD98059. BDNF-induced survival was only blocked by Ly294002 and BDNF-induced proliferation was only inhibited by PD98059. CONCLUSION: BDNF promotes angiogenesis of HUVECs in vitro. The ERK and Akt are two crucial events in BDNF-mediated signal transduction leading to HUVECs angiogenesis by different mechanisms. Moreover, the latter is more important.     

Effects of cinnamyl aldehyde on c-Fos and c-Myc expression in NIH3T3 cells

AIM: Cinnamyl aldehyde (CA) is one alcohol ingredient derived from Cinnamomum cassia,which is widely used in treating chronic skin wound in Chinese medicine with the curative effect of ‘rescuing YANG’.The purpose of the present study was to investigate the expression of c-Fos,c-Myc proteins at different time points in NIH3T3 treated with CA and explore the possible mechanism of promoting cell proliferation by CA.METHODS: MTT assay was used for observing cell proliferation.Expression of c-Fos and c-Myc proteins in NIH3T3 cells were assessed by immunocytochemistry assay.RESULTS: The cell proliferation was promoted obviously when CA concentration was between 8.8×10^-2 μg/L and 8.8×10 μg/L.CA at concentration of 5.5 μg/L significantly induced expression of c-Fos,c-Myc proteins at 2-3 h after the stimulation compared with control group (P<0.01).CONCLUSION: CA increases expression of c-Fos and c-Myc proteins,which may be one of mechanisms for CA to promote NIH3T3 cell proliferation.     

Effect of formononetin on viability,migration and invasion of ovarian cancer cells in vitro

AIM To observe the effect of formononetin on the viability, migration and invasion of ovarian cancer cells, and to explore its mechanism. METHODS Human ovarian serous cystadenocarcinoma SKOV-3 cells were cultured in vitro. The cells were treated with formononetin at 0, 25, 50 and 100 μmol/L for 48 h. The cell viability was measured by MTS assay. The migration and invasion abilities of the SKOV-3 cells were detected by scratch wound assay and Transwell assay. RT-qPCR and Western blot were used to detect the mRNA and protein levels of E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS The viability of SKOV-3 cells was decreased with the increase in the formononetin concentration compared with control group (P<0.01). The wound migration distance of the cells in 50 μmol/L formononetin group was less than that in control group (P<0.01). The number of invasive SKOV-3 cells across the Transwell sub-compartment was significantly decreased in 50 μmol/L formononetin group compared with control group (P<0.01). The mRNA and protein levels of E-cadherin in 50 μmol/L formononetin group were significantly higher than those in control group (P<0.01), while the mRNA and protein levels of MMP-9 in 50 μmol/L formononetin group were significantly lower than those in control group (P<0.01). CONCLUSION Formononetin inhibits the migration and invasion abilities of ovarian cancer SKOV-3 cells by increasing expression of E-cadherin and decreasing expression of MMP-9.     

Effect of rhEGF activating extracellular signal-regulated kinase on the expression of Bcl-2 and p53 protein in human amniotic cells

AIM: To explore the effect of recombinant human epidermal growth factor (rhEGF) on the ERK1/2 pathway and its safety in use when the proliferation of cultured FL cells (human amnion epithelial cells) were promoted by rhEGF.METHODS: FL cells were stimulated by rhEGF at various concentrations.The proliferation rate of FL cells was evaluated by MTT assay.Western blotting was used to detect phosphorylation of ERK1/2 (p-ERK1/2) and the change of Bcl-2 and P53 protein.RESULTS: The proliferation rate FL cells reached the maximum when the concentration of rhEGF was 10 μg/L (42.4％，P<0.01).Western blotting showed that the activity of p-ERK1/2 increased significantly when rhEGF ranged in 10 μg/L to 60 μg/L.Level of Bcl-2 was unchanged in each groups.Level of P53 decreased significantly when the concentration of rhEGF was 60 μg/L.CONCLUSION: It is safe when rhEGF presents in optimization concentration of 10 μg/L.The capability of apoptosis may be depressed when rhEGF is in larger dose.