In vitro anticancer property of a novel thalidomide analogue through inhibition of NF-κB activation in HL-60 cells

Aim： To investigate the anticancer property and possible mechanism of action of a novel sugar-substituted thalidomide derivative （STA-35） on HL-60 cells in vitro.
Methods： TNF-α-induced NF-κB activation was determined using a reporter gene assay. The MTT assay was used to measure cytotoxicity of the compound. The appearance of apoptotic Sub-G1 cells was detected by flow cytometry analysis. PARP cleavage and protein expression of NF-κB p65 and its inhibitor IκB were viewed by Western blotting.
Results： STA-35 （1-20 μmol/L） suppressed TNF-α-induced NF-κB activation in transfected cells （HEK293/pNiFty-SEAP） in a dose- （1-20 μmol/L） and time-dependent （0-48 h） manner. It was also shown that STA-35 exerted a dose-dependent inhibitory effect on HL-60 cell proliferation with an IC50 value of 9.05 lamol/L. In addition, STA-35 induced apoptosis in HL-60 cells, as indicated by the appearance of a Sub-G1 peak in the cell cycle distribution, as well as poly ADP-ribose polymerase （PARP） cleavage. Subsequently, both NF-κB p65 and its inhibitor IκB gradually accumulated in cytoplasmic extracts in a dose- and time-dependent manner, indicating the blockage of NF-κB translocation induced by TNF-α from the cytoplasm to the nucleus.
Conclusion： A novel sugar-substituted thalidomide derivative, STA-35, is potent toward HL-60 cells in vitro and induces apoptosis by the suppression of NF-κB activation.     

在患有急性肺损伤的小猪的肺泡巨噬细胞中一氧化氮，表面活性剂和糖皮质激素对核因子-κB和激活蛋白-1的活性的调控作用

AIM： To investigate whether acute lung injury (ALI) in ventilated piglets with bacterial infection affects NF-κB and AP-1 expression in alveolar macrophages (AM) and whether nitric oxide (NO), surfactant (Surf), glucocorticoids (GC) affect NF-κB and AP-1 activation in AM in vivo and in vitro. METHODS: The animals were intraperitoneally injected Escherichia coli, which caused ALI. Nuclear extracts of AM were analyzed by electrophoretic mobility shift assay (EMSA) for the nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) expression. Detection of IκB-α protein was from cytoplasmic extract by Western blotting. Immunocytochemistry staining was used for intracellular location of p65 subunits of NF-κB. RESULTS: In ex vivo experiments, strikingly higher expression of NF-κB and AP-1 by EMSA was found 6h after bacterial injection in contrast to the Normal group. In the NO, SNO,and GC groups, markedly attenuated NF-κB and AP-1 activation was observed. The NF-κB and AP-1 activation in Surf group showed lower levels of the expression. Immunoblotting of AM cytoplasmic extract showed low expression of IκB-α protein in the Control and Surf groups. The stronger expression was observed in the NO, GC,and SNO groups. AM of the Control and Surf groups showed intense nuclear staining, with decreased nuclear staining in the NO, GC and SNO groups. In in vitro experiment, it caused a significant increase in NF-κB and AP1 activity in AM 1h after exposure to lipopolysaccharides (LPS). In AM treated by LPS SNP and LPS GC, all showed decrease of DNA binding activity of NF-κB and AP-1 compared to those exposed to LPS Surf. Immunoblotting of AM cytoplasmic extract showed that LPS stimulation of AM resulted in the low expression of IκB-α protein, which was not observed in the presence of SNP and methylprednisolone. However, the surfact antdid not show such effect. LPS Surf-exposed AM had intense nuclear staining, whereas decreased nuclear staining in the LPS NO and LPS GC-treated cultures was found, confirming a decrease in NF-rd3 activity. CONCLUSION:Activation of NF-κB was found in AM of ventilated piglets with bacterial ALI. NO and GS could prevent NF-κB and AP-1 activation in vivo and in vitro. Surfactant has limited effects on NF-κB and AP-1 activity.     

Effect of α-pinene on nuclear translocation of NF-κB in THP-1 cells

AIM: To study the effects of α-pinene on nuclear translocation of nuclear factor-κB (NF-κB) and the expression of the inhibitor of NF-κB (IκBα) in human monocyte THP-1 cell line. METHODS: THP-1 cells were incubated with α-pinene (1, 10, and 100 mg/L, for 30 min) before being stimulated with lipopolysaccharide (LPS, 1 mg/L, 30 min).The location of NF-κB p65 subunit (NF-κB/p65) in THP-1 cells was detected by immunofluorescence and laser scanning confocal microscope (LSCM). The expression of NF-κB/p65 in nuclei and that of IκBα in cytoplasm were measured by Western-blot analysis. RESULTS: The majority of FITC-labelled NF-κB/p65 was located in the nuclei being stimulated with LPS. Whereas, no such fluorescence was seen in the nuclei of the groups pretreated with α-pinene or control cells, α-Pinene pretreatment decreased the NF-κB/p65 nuclear translocation in LPS-stimulated THP-1 cells, and this effect was dose-dependent, but there was no reaction in LPS-unstimulated THP-1 cells, α-Pinene pretreatment increased IκBα protein level in cytoplasm, compared with that in LPS-stimulated THP-1 cells. CONCLUSION: In a dose-related fashion, α-pinene inhibits the nuclear translocation of NF-κB induced by LPS in THP- 1 cells, and this effect is partly due to the upregulation of IκBα expression.     

Daidzein affects proliferation and apoptosis in non-small cell lung cancer cells:role of p53 signaling pathway北大核心CSCD

Aim To investigate the effects of daidzein（DD） on the proliferation and apoptosis of non-small cell lung cancer cells,with a focus on the possible role of the p53 signaling pathway in this regard. Methods CCK-8 method and flow cytometry were used to detect the effects of soy isoflavone crude extract and DD on the viability and apoptosis of HELF and H1299 cells. Gene microarray was used to detect the changes in gene expression after treatment of H1299 cells with DD. GSEA and differential analysis were used to screen the major pathways and key genes. RT-qPCR and Western blot were performed to verify the differences in mRNA and protein expression of key genes（p53 and CASP9） in the major pathways. After p53 inhibitor Pifithrin-α inhibited the expression of p53,the effect of DD on p53 mRNA and protein expression levels was examined,and the proliferative effect on H1299 cells was observed. Results Soy isoflavone crude extract and DD promoted proliferation and inhibited apoptosis of normal lung cells and inhibited proliferation and promoted apoptosis of lung cancer cells. p53 signaling pathway was significantly enriched in the DD-treated group（NES=1.78,P=0.000）,and the expressions of p53 and CASP9 genes were found to be significantly up-regulated in the treated group. Compared with the control group,mRNA expression of CASP9 and p53 significantly increased in both HELF and H1299 cells treated with DD（P<0.05）,and p53 protein expression also increased in HELF cells（P<0.05）. After inhibition of p53 expression,DD significantly increased the mRNA expression of p53 in H1299 and HELF cells（P<0.05） and also markedly increased the expression of p53 protein in H1299 cells（P<0.05）,and it was observed that DD inhibited the proliferation of lung cancer cells. Conclusions DD inhibits the proliferation and promotes the apoptosis of lung cancer H1299 cells,and the mechanism mainly involves the p53 signaling pathway. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Inhibitory effect of 1,8-cineol （eucalyptol） on Egr-1 expression in lipopolysaccharide-stimulated THP-1 cells

Aim： To study the effects of 1,8-cineol （eucalyptol） on the expression of early growth response factor-1 （Egr-1） and NF-κB in the human monocyte THP-1 cell line stimulated by lipopolysaccharide （LPS）. Methods： The THP-1 cells were incu- bated with serial doses of 1,8-cineol （1, 10, and 100 mg/L, 30 min） before being stimulated with LPS （1 mg/L, 30 min）. The localization of Egr-1 in the THP-1 cells was detected by immunofluorescence and a laser scanning confocal microscope. The expression of Egr-1 in the nuclei and whole cell, and NF-κB in the nuclei, were measured by Western blot analysis. Results： When stimulated by LPS, the FITC- labeled Egr-1 was detected mainly in the nuclei. Moreover, the expression of Egr-1 in the whole cell increased markedly compared with the control cells. 1,8- Cineol pretreatment decreased the expression of Egr-1 in both the nuclei and whole cell of the LPS-stimulated THP- 1 cells, and this effect was concentration- dependent, but there was no reaction on the expression of NF-κB in the nuclei protein in the LPS-stimulated THP-1 cells. Conclusion： In a concentration-depen- dent manner, 1,8-Cineol reduces LPS-induced Egr-1 expression in nuclei and in whole cell of THP-1 cells, but shows no effect on NF-κB expression.     

Effect of simvastatin on endothelium-dependent vaso-relaxation and endogenous nitric oxide synthase inhibitor

AIM: To investigate the effect of simvastatin on endothelium-dependent vasorelaxation and endogenous nitric oxide synthesis inhibitor asymmetric dimethylarginine (ADMA) in rats and cultured ECV304 cells. METHODS: Endothe-lial injury was induced by a single injection of low density lipoprotein (LDL) (4 mg/kg, 48 h) in rats or incubation with LDL (300 mg/L) or oxidative-modified LDL (100 mg/L) in cultured ECV304 cells, and vasodilator responses to acetylcholine (ACh) in the aortic rings and the level of ADMA, nitrite/nitrate (NO) and tumor necrosis factor-alpha (TNF-α) in the serum or cultured medium were determined. And the adhesion of the monocytes to endothe-lial cells and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the cultured ECV304 cells were measured. RESULTS: A single injection of LDL decreased endothelium-dependent relaxation to ACh, markedly increased the serum level of endogenous ADMA and TNF-α, and reduced serum level of NO. Pretreatment with simvastatin (30 or 60 mg/kg) markedly attenuated inhibition of vasodilator responses to ACh, the increased level of TNF-α and the decreased level of NO by LDL, but no effect on serum concentration of endogenous ADMA. In cultured ECV304 cells, LDL or ox-LDL markedly increased the level of ADMA and TNF-α and potentiated the adhesion of monocytes to endothelial cells, concomitantly with a significantly decrease in the activity of DDAH and serum level of NO. Pretreatment with simvastatin (0.1, 0.5, or 2.5 μmol/L) markedly decreased the level of TNF-α and the adhesion of monocytes to endothelial cells, but did not affect the concentration of endogenous ADMA and the activity of DDAH. CONCLUSION: Simvastatin protect the vascular endothelium against the damages induced by LDL or ox-LDL in rats or cultured ECV304 cells, and the beneficial effects of simvastatin may be related to the reduction of inflammatory cytokine TNF-α level.     

Dauricine inhibits insulin-like growth factor-Ⅰ-induced hypoxia inducible factor la protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim： To investigate the effects of dauricine （Dau） on insulin-like growth factor-Ⅰ （IGF-Ⅰ）-induced hypoxia inducible factor 1α （HIF-1α） and vascular endothelial growth factor （VEGF） expression in human breast cancer cells （MCF-7）. Methods： Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-Ⅰ for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell （HUVEC） tube formation assay. An in vitro invasion assay on HUVECs was performed. Results： Dau significantly inhibited IGF-Ⅰ-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-Ⅰ. Mechanistically, Dau suppressed IGF-Ⅰ-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-Ⅰ-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-Ⅰ-induced invasion of HUVECs. Conclusion： Dau inhibits human breast cancer angiogenesis expression, which may provide a novel potential mechanism for by suppressing HIF-1α protein accumulation and VEGF the anticancer activities of Dau in human breast cancer.     

BMP6 reverses TGF-β1-induced changes in HK-2 cells： implications for the treatment of renal fibrosis

Aim： The aim of the study was to investigate the potential role of BMP6 in TGF-β1-mediated changes in HK-2 cells.
Methods： BMP6 was purified via heparin affinity and reverse phase liquid chromatography. The purity, specificity, and bioactivity of BMP6 were determined by SDS-PAGE, Western blot assays, and the induction of alkaline phosphatase （ALP） activity, respectively. Cell proliferation, morphology, and expression levels of α-SMA and E-cadherin were assessed by cell viability, microscopy, and Western blot assays, respectively. In addition, cell adhesion abilities were determined by counting the number of attached cells, The expression of fibroneetin, collagen Ⅳ, matrix metalloproteinases 2 （MMP-2）, and tissue inhibitors of matrix metalloproteinases 2 （TIMP-2） were analyzed using RT-PCR. MMP-2 activity was analyzed by zymography, whereas the activation of the MAPKs and Smad signaling were analyzed using Western blot assays and a reporter gene assay, respectively.
Results： Our results indicated that recombinant BMP6 induced ALP activity Jn a dose-dependent and time-course-dependent manner. Treatment with TGF-β1 reduced both the cell proliferation and the expression of E-cadherin, induced a morphological transformation, decreased the expression and activity of MMP-2, and increased the expression levelsof α-SMA, flbronectin, and TIMP-2 in HK-2 cells. All of these effects were inhibited when cells were treated with TGF-β1 in combination with rhBMP6, whereas rhBMP6 alone demonstrated no such effect. Treatment with TGF-β1, rhBMP6, or a combination of both had no effect on the expression of collagen IV. In addition, the administration of rhBMP6 prevented the enhanced adhesion behavior triggered by TGF-β1. Furthermore, the addition of rhBMP6 abrogated the JNK and Smad2/3 signaling that was activated by TGF-β1.
Conclusion： BMP6 ameliorated the TGF-131-induced changes in HK-2 cells. The suppression of TGF-β1-mediated JNK and Smad2/3 signaling activation were implicated in these effect     

Preliminary molecular basis of Danggui-buxue-tang on Qi deficiency and blood stasis syndrome

Objective To study the molecular biology mechanism of Danggui-buxue-tang on tonifying Qi and controlling blood circulation through modulation the immune functional genes.Methods Rats were randomly divided into control group,model group,and Danggui-buxue-tang group.Effect of Dang-guibuxue-tang on mRNA expressions of IL-1β,TNF-α,HSP-70,NF-κB,p38MAPK and JNK in blood cells of rats with Qi deficiency and blood stasis were measured with real-time fluorescent quantitative-PCR at 5th,14th and 28th day.And that in artery wall were determined at the 28th day.NF-κB/p65 and p-c-jun protein expressions in rat artery wall were detected by western blotting as well.Results In model group,TNF-α,IL-1β,NF-κB,HSP-70,p38MAPK and JNK mRNA expression in blood cell increased significantly compared with control group.Compared with model group,mRNA expression of IL-1βand JNK decreased significantly;TNF-α decreased at 5th,14th day;HSP-70,p38MAPK decreased at 14th,28th day;NF-κB decreased at 28th day.In model group,TNF-α,IL-1β,NF-κB,p38MAPK and JNK mRNA expression in artery increased compared with control group,excluded HSP-70,and that in Danggui-buxue-tang group decreased significantly.In model group,NF-κB/p65 and p-c-jun protein expression in artery increased compared with control group,and that in Danggui-buxue-tang group decreased significantly.Conclusions The effects of Dang-guibuxue-tang on Qi deficiency and blood stasis syndrome was brought from the regulating of cytokine network at multi-link and multi-target,NF-κB,p38MAPK and JNK signal transduction pathways included.Through which the immune system and whole body reached to a functional balance status.     

Compound FLZ inhibits lipopolysaccharide-induced inflammatory effects via down-regulation of the TAK-IKK and TAK-JNK/ p38MAPK pathways in RAW264.7 macrophages

Aim： The aim of this study was to investigate the effect of the squamosamide derivative FLZ （N-2-（4-hydroxy-phenyl）-ethyl-2-（Z,5-dimethoxy-phenyl）-3-（3-methoxy-4-hydroxy-phenyl）-acrylamide） on lipopolysaccharide （LPS）-induced inflam-matory mediator production and the underlying mechanism in RAW264.7 macrophages. Methods： RAW264.7 cells were preincubated with non-toxic concentrations of compound FLZ （1, 5, and 10 μmol/L） for 30 min and then stimulated with 10 μg/L LPS. The production of nitric oxide （NO）, the expression of inducible nitric oxide synthase （iNOS） and cyclooxygenase 2 （COX-2）, and the activation of nuclear factor kappa-B （NF-KB） and mitogen-activated protein kinase （MAPK） pathways were examined. Results： FLZ significantly inhibited the LPS-induced production of NO, as well as the expression of iNOS and COX-2 at both the RNA and the protein levels in RAW264.7 cells. The LPS-induced increase in the DNA binding activity of NF-KB and activator protein i （AP-1）, the nuclear translocation of NF-κB p65, the degradation of the inhibitory κBα protein （IκBα） and the phosphorylation of IκBα, IκB kinase （IKK） α/β, c-Jun NH2-terminal kinase （JNK） and p38 MAPKs were all suppressed by FLZ. However, the phosphorylation of extracellular signal-regulated kinase （ERK） was not affected. Further study revealed that FLZ inhibited the phosphorylation of transforming growth factor-β （TGF-β）-activated kinase 1（TAK1）, which is an upstream signaling molecule required for IKKα/β, JNK and p38 activation. Conclusion： FLZ inhibited the LPS-induced production of inflammatory mediators at least partly through the downregulation of the TAK-IKK and TAK-JNK/p38MAPK pathways.     

TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways

Aim： Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-a has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro. Methods： Human umbilical vein endothelial cells （HUVECs） were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-a-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTTassay. Monocyte migration was measured with transwell migration assay. Results： Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α （2, 5 ng/mL） induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α （5 ng/mL） caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor （SP600125）, p38 MAPK inhibitor （SB202190） or PI-3K inhibitor （LY294002） significantly suppressed TNF-a-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-a-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone （up to 500 nmol/L） failed to affect TNF-a-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-a-induced CXCL1 from HUVECs attracted human monocyte migration. Conclusion： TNF-a stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.     

Heme oxygenase-1 mediates the anti-inflammatory effect of isoflurane preconditioning in LPS-stimulated macrophages

Aim： The aim of this study was to investigate the anti-inflammatory action of isoflurane preconditioning in a model of lipopolysaccharide （LPS）-induced inflammation in RAW 264.7 macrophages and examine the role of heme oxygenase （HO）-1 in this process. Methods： Murine 264.7 macrophages were pretreated with or without 1%-3% isoflurane for 1 h. Thirty minutes later, the cells were incubated with or without LPS for 24 h. Cell viability was assessed using a 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay and cell injury was assessed by measuring the release of lactate dehydrogenase （LDH）. HO-1 and inducible nitric oxide synthase （iNOS） protein expression was analyzed by Western blotting. Tumor necrosis fac- tor （TNF）-a levels, nitrite production and HO activity were also determined. Results： Pretreatment with the nontoxic and clinically approved anesthetic isoflurane potently attenuated the cell injury and the decrease in cell viability that was induced by LPS. Treatment or pretreatment with 2% isoflurane induced HO-1 protein expression and caused an induction of HO activity. This result correlated with a decrease in iNOS expression, a decrease in the production of nitric oxide （NO） and impaired release of TNF-α in LPS-stimulated macrophages. Blockade of HO activity with tin protoporphyrin （SnPP） reversed these effects. Conclusion： Isoflurane preconditioning exerts its anti-inflammatory activity through the HO-1 pathway in an in vitro inflammation model.     

Puerarin inhibits angiotensin II-induced cardiac hypertrophy via the redox-sensitive ERK1/2, p38 and NF-κB pathways

Aim： To investigate the effects of puerarin （Pue）, an isoflavone derived from Kudzu roots, on angiotensin II （Ang II）-induced hypertrophy of cardiomyocytes in vivo and in vitro.
Methods： C57BL/6J mice were infused with Ang II and treated with Pue （100 mg·kg-1·d-1, po） for 15 d. After the treatment, systolic blood pressure （SBP） and left ventricular wall thickness were assessed. The ratios of heart weight to body weight （HW/BW） and left ventricular weight to body weight （LVW/BW） were determined, and heart morphometry was assessed. Expression of fetal-type genes （ANP, BNP and β-MHC） in left ventricles was measured using semi-quantitative RT-PCR. Mouse primary cardiomyocytes were treated with Pue （50, 100, 200 μmol/L）, then exposed to Ang II （1 μmol/L）. ROS level was examined with flow cytometry, the binding activity of NF-κB was determined using EMSA. Western blot was used to measure the levels of ERK1/2, p38 and NF-κB pathway proteins. [3H]leucine incorporation was used to measure the rate of protein synthesis.
Results： Oral administration of Pue significantly suppressed Ang II-induced increases in the myocyte surface area, HW/BW, LVW/BW, SBP and left ventricular wall thickness. Furthermore, Pue significantly suppressed Ang II-induced increases in ANP, BNP and β-MHC expression in the left ventricles in vivo. Treatment of cardiomyocytes with Pue （50–500 μmol/L） did not affect the viability of cardiomyocytes in vitro. Pretreatment of cardiomyocytes with Pue dose-dependently inhibited Ang II-induced increases in ROS production, NF-κB binding activity, protein synthesis and cell breadth. Furthermore, pretreatment with Pue significantly suppressed Ang II-induced activation of ERK1/2, p38 and the NF-κB pathway proteins and the expression of ANP and β-MHC in cardiomyocytes. The positive drug valsartan exerted similar effects on Ang II-induced cardiac hypertrophy in vivo and in vitro.
Conclusion： Pue attenuates Ang II-induced cardiac hypertrophy by inhibiting activation of the redox-sensitive ERK1/2, p38 and the NF-κB pathways.     

Insulin-like growth factor-1 promotes cell cycle progression via upregulation of cyclin D1 expression through the phosphatidylinositol 3-kinase/nuclear factor-κB signaling pathway in FRTL thyroid cells

Aim： Insulin-like growth factor-1 （IGF-1） is an important hypertrophic and cell cycle progression factor for a number of cell types. It has been proven that IGF-1 is involved in the regulation of thyroid proliferation and cell cycle progression; however, the exact mechanism of this regulation has not been fully elucidated. In the present study, we investigated the effect of IGF-1 on the expression of cyclin D1, an important cell cycle regulatory protein, and a signaling pathway involved in IGF-1＇s effect on cyclinD1 expression in FRTL thyroid cells.
Methods： FRTL thyroid cells were treated with IGF-1 or vector control for 24 h. As appropriate to individual experiments, a phosphatidylinositol 3-kinase （PI3K） inhibitor, LY294002, and/or a nuclear factor-κB （NF-κB） inhibitor, BAY11-7082, were added 1 h prior to IGF-1 treatment. Western blotting was used to detect cyclin D1 protein expression. Immunofluorescence was performed to analyze the expression of IκBα, an NF-κB inhibitory protein. Cell cycle analysis was performed by fluorescence activated cell sorting （FACS）.
Results： IGF-1 increased the cyclin D1 expression in thyroid cells. This increase was blocked by pretreatment with LY294002 or BAY11-7082. Further studies showed that IGF-1 specifically induced NF-κB activity. Treatment with IGF-1 could accelerate cell cycle progression from G0/G1 to S phase, whereas this progression was inhibited by the presence of LY294002 or BAY11-7082.
Conclusion： In summary, the results of the present study show that in FRTL cells, IGF-1 promotes cell cycle progression via an upregulation of cyclin D1 expression, at least partially through the PI3K/NF-κB signaling pathway.     

Salvianic acid A inhibits induction of inflammatory mediators by blocking Nuclear Factor-κB activation in macrophages

Objective To investigate the anti-inflammation effect and possible mechanism of Salvianic acid A(SAA)in mouse peritoneal macrophages.Methods Peritoneal macrophages were obtained from BALB/c mice.LPS induced nitric oxide(NO),tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in supernatant,protein expression of inducible nitric oxide synthase(iNOS),matrix metalloproteinase-9(MMP-9)and activation of nuclear factor-kappa B(NF-κB)in the extract were measured.Results SAA strongly inhibited the excessive production of NO,TNF-α and IL-6 in LPS-induced peritoneal macrophages in a concentration-dependent manner and blocked the expression of iNOS and MMP-9.Treatment with LPS alone increased the translocation of NF-κB(p65)from cytosol to the nucleus,but the SAA inhibited the translocation of NF-κB(p65).Conclusions The results showed that SAA had strong anti-inflammatory effects in LPS-stimulated peritoneal macrophages.The important mechanism is due to its inhibition of NF-κB activation.     

Cysteinyl leukotriene receptor I mediates LTD4- induced activation of mouse microglial cells in vitro

Aim： To investigate the roles of cysteinyl leukotriene receptors CysLT1R and CysLT2R in leukotriene D4 （LTD4）-induced activation of microglial cells in vitro.
Methods： Mouse microglial cell line BV2 was transfected with pcDNA3.1（＋）-hCysLT1R or pcDNA3.1（＋）-hCysLT2R. The expression of relevant mRNAs and proteins in the cells was detected using RT-PCR and Western blotting, respectively. Phagocytosis was determined with flow cytometry analysis. The release of interleukin-1β （IL-1β） from the cells was measured using an ELISA assay.

Results： The expression of CysLT1R or CysLT2R was considerably increased in the transfected BV2 cells, and the receptors were mainly distributed in the plasma membrane and cytosol. Treatment of the cells expressing CysLT1R or CysLT2R with CysLT receptor agonist LTD4 （0.1–100 nmol/L） concentration-dependently enhanced the phagocytosis, and increased mRNA expression and release of IL-1β. Moreover, the responses of hCysLT1R-BV2 cells to LTD4 were significantly larger than those of hCysLT2R-BV2 or WT-BV2 cells. Pretreatment of hCysLT1R-BV2 cells with the selective CysLT1R antagonist montelukast （1 μmol/L） significantly blocked LTD4-induced phagocytosis as well as the mRNA expression and release of IL-1β, whereas the selective CysLT2R antagonist HAMI 3379 （1 μmol/L） had no such effects.

Conclusion： CysLT1R mediates LTD4-induced activation of BV2 cells, suggesting that CysLT1R antagonists may exert anti-inflammatory activity in brain diseases.     

Regulation of activity of nuclear factor-kB and activator protein-1 by nitric oxide, surfactant and glucocorticoids in alveolar macrophages from piglets with acute lung injury

AIM: To investigate whether acute lung injury (ALl) in ventilated piglets with bacterial infection affects NF-κB and AP-1 expression in alveolar macrophages (AM) and whether nitric oxide (NO), surfactant (Surf), glucocorticoids (GC) affect NF-κB and AP-1 activation in AM in vivo and in vitro. METHODS: The animals were intraperitoneally injected Escherichia coli, which caused ALl. Nuclear extracts of AM were analyzed by electrophoretic mobility shift assay (EMSA) for the nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) expression. Detection of IκB-α protein was from cytoplasmic extract by Western blotting. Immunocytochemistry staining was used for intracellular location of p65 subunits of NF-κB. RESULTS: In ex vivo experiments, strikingly higher expression of NF-κB and AP-1 by EMSA was found 6 h after bacterial injection in contrast to the Normal group. In the NO, SNO, and GC groups, markedly attenuated NF-κB and AP-1 activation was observed. The NF-κB and AP-1 activation in Surf g