Inhibitory Effects of Parthenolide on the Angiogenesis Induced by Human Multiple Myeloma Cells and the Mechanism

The inhibitory effects of parthenolide （PTL） on angiogenesis induced by multiple myeloma （MM） cells in vitro and the mechanism were investigated. Human MM line RPMI8226 cells were cultured in vitro. The effects of MM culture supernatant on the migration and tubule formation ability of human umbilical vein endothelial cells （HUVECs） treated with PTL were observed. By using Western blot, the expression of p65 and IкB-α in MM cells was detected. RT-PCR was used to assay the expression of VEGF, IL-6, MMP2 and MMP9 mRNA in MM cells. ELISA was used to measure the levels of VEGF and IL-6 in MM cell culture supernatant. The expression of MMP2 and MMP9 in MM cells was examined by immunohistochemistry. （1） In 3.5, 5.0, 7.5 and 10 μmol/L PTL groups the number of migrated cells was 310±56, 207±28, 127±21 and 49±10 respectively, which was significantly different from that in positive control group （598±47） （P〈0.01）. In 3.5 and 5.0 μmol/L PTL groups the areas of capillary-like structures were 0.092±0.003 and 0.063±0.002 mm2, significantly less than in positive control group （0.262±0.012 mm2） （P〈0.01）, but in 7.5 and 10 μmol/L PTL groups no capillary-like structures were found; （2） After treatment with different concentrations of PTL for 48 h, the expression of p65 protein was gradually decreased, while that of IкB-α was gradually enhanced with the increased concentration of PTL; （3） After treatment with 3.5, 5.0, 7.5 and 10 μmol/L PTL for 48 h, the VEGF levels in the supernatant were 2373.4±392.2, 1982.3±293.3, 1247.0±338.4 and 936.5±168.5 pg/mL respectively, significantly different from those in positive control group （2729±440.0 pg/mL） （P〈0.05）. After treatment with 7.5 and 10 μmol/L PTL, the IL-6 levels in the culture supernatant were 59.6±2.8 and 41.4±9.8 pg/mL respectively, signifi- cantly lower than in positive control group （1287.3±43.5 pg/mL） （P〈0.05）; （4） RT-PCR revealed that PTL could significantly inhibit the expression of V     

Activation of NF-κB in bronchial epithelial cells from children with asthma

Objectives To determine whether nuclear factor-κB (NF-κB) is activated in epithelial cells from children with asthma and to understand the role of NF-κB in airway inflammation in asthma. Methods Bronchial mucosa specimens were obtained from 9 children with asthma and 6 control subjects. NF-κB expression in epithelial cells were detected by immunohistochemical examination, and NF-κB-DNA binding was measured by electrophoretic mobility shift assay (EMSA). Results Nuclear expression of NF-κB in epithelial cells was observed in the 9 asthmatic children. NF-κB-DNA binding was found in 4 asthmatic children (EMSA was performed in 6 asthmatic children). In contrast, both nuclear expression and NF-κB-DNA binding were absent in the 6 control subjects. Conclusion These results indicated that NF-κB is activated in epithelial cells from asthmatic children and the NF-κB activation may be the basis for the increased expression of many inflammatory genes and for airway inflammation in asthma.     

Influence of silencing TRAF6 with shRNA on LPS/TLR4 signaling <Emphasis Type="Italic">in vitro</Emphasis>

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide(LPS)/toll-like receptor(TLR)-4 signaling pathway in vitro.Four plasmids(pGCsi-TRAF6-shRNA1,2,3,4) containing different shRNA sequences were designed and synthesized.The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay.Inflammatory cellular models were established by LPS stimulation.Levels of TNF-α,IL-1β and TGF-β1 in the supernatants,mRNA expressions of TRAF6,IL-6 and COX-...     

Effects of Triptolide on the Expression and Activity of NF-κB in Synovium of Collagen-induced Arthritis Rats

Summary： The expression and activity of NF-kB in the synovium of collagen-induced arthritis （CIA） rats was detected in order to investigate the possible therapeutic effects of triptolide on rheumatoid arthritis （RA）. The experimental Wistar rat model of CIA was set up by intradermal injection of emulsion of bovine collagen 11 and the successful rate of setting-up models was evaluated by arthritis index （AI）. Rats were grouped randomly into three groups： normal, model and treatment group. The expression of TNF-α and IL-6 in synovial fluid was detected by ELISA, and the expression and activity of NF kB in synovium by immunohistochemistry method and by electrophoretic mobility shift assay （EMSA） respectively. As compared with normal group, the expression of TNF a and IL-6 in synovia （P〈0. 05）, and the expression and activity of NF-kB （P〈0.05） in synovium were increased in model group. There was statistical difference in above-mentioned indexes between model group and treatment group. Triptolide may play a protective role in IRA via downregulating the expression and activity of NF-kB in synovium.     

Effects of andrographolide on the activation of mitogen activated protein kinases and nuclear factor-κB in mouse peritoneal macrophage-derived foam cells

Objective:To observe the effect of andrographolide on the activation of mitogen-activated protein kinases(MAPKs) and expression of nuclear factor-κB(NF-k B) in macrophage foam cells.Methods:The mouse peritoneal macrophages were cultured in the media in the presence of oxidized low-density lipoprotein (ox-LDL),ox-LDL+andrographolide,or neither(control).The phosphorylation of MAPK molecules(p38MAPK, JNK,ERK1/2) and the expressions of NK-κB p65 were examined by Western blot.Results:As compared with cells in the control group,the expressions of phospho-p38 and NF-κB p65 were increased in the cells cultured with either ox-LDL or ox-LDL+andrographolide(P<0.01),but attenuated significantly in the presence of ox-LDL+ andrographolide when compared with ox-LDL(P<0.05).The phospho-JNK increased in the presence of either ox-LDL or ox-LDL+andrographolide when compared with control cells(P<0.01),but no significant difference existed between ox-LDL and ox-LDL+andrographolide(P>0.05).The expression of phospho-ERK1/2 was increased in the presence of ox-LDL compared with the control cells(P<0.01),but no significant differences existed between the cells cultured in the presence of ox-LDL+andrographolide and the control medium(P>0.05). Conclusions:Andrographolide could inhibit the activation of ERK1/2,p38MAPK and NK-k B induced by ox-LDL in macrophage foam cells,which might be one of its mechanisms in preventing atherosclerosis.     

Inhibitory Effect of Oxymatrine on Quartz-induced Secretion of TNF-α by the Pulmonary Alveolar Macrophages in the Fibroblast Proliferation

The massive proliferation of pulmonary fibroblasts is the common consequence shared by lung diseases. The over-expression of TNF-α plays an important role in the proliferation of pulmonary fibroblasts[1, 2]. Under patho-logical conditions, TNF-α is predominantly secreted by activated alveolar macrophages of lung. The high level of TNF-α acts on the receptors on the cell membrane of fibroblasts, mediates the conduction of intracellular sig-nals and eventually activates the nuclear factor…     

Extract of Fructus Schisandrae chinensis Inhibits Neuroinflammation Mediator Production from Microglia via NF-κB and MAPK Pathways

Objective: To investigate the anti-neuroinflammation effect of extract of Fructus Schisandrae chinensis(EFSC) on lipopolysaccharide(LPS)-induced BV-2 cells and the possible involved mechanisms. Methods: Primary cortical neurons were isolated from embryonic(E17-18) cortices of Institute of Cancer Research(ICR) mouse fetuses. Primary microglia and astroglia were isolated from the frontal cortices of newborn ICR mouse. Different cells were cultured in specific culture medium. Cells were divided into 5 groups: control group, LPS group(treated with 1 μg/mL LPS only) and EFSC groups(treated with 1 μg/mL LPS and 100, 200 or 400 mg/mL EFSC, respectively). The effect of EFSC on cells viability was tested by methylthiazolyldiphenyltetrazolium bromide(MTT) colorimetric assay. EFSC-mediated inhibition of LPS-induced production of pro-inflammatory mediators, such as nitrite oxide(NO) and interleukin-6(IL-6) were quantified and neuron-protection effect against microglia-mediated inflammation injury was tested by hoechst 33258 apoptosis assay and crystal violet staining assay. The expression of pro-inflammatory marker proteins was evaluated by Western blot analysis or immunofluorescence. Results: EFSC(200 and 400 mg/mL) reduced NO, IL-6, inducible nitric oxide synthase(iN OS) and cyclooxygenase 2(COX-2) expression in LPS-induced BV-2 cel s(P0.01 or P0.05). EFSC(200 and 400 mg/mL) reduced the expression of NO in LPS-induced primary microglia and astroglia(P0.01). In addition, EFSC al eviated cel apoptosis and inflammation injury in neurons exposed to microglia-conditioned medium(P0.01). The mechanistic studies indicated EFSC could suppress nuclear factor(NF)-κB phosphorylation and its nuclear translocation(P0.01). The anti-inflammatory effect of EFSC occurred through suppressed activation of mitogen-activated protein kinase(MAPK) pathway(P0.01 or P0.05). Conclusion: EFSC acted as an anti-inflammatory agent in LPS-induced glia cel s. These effects might be realized through blocking of NF-κB activity and inhibition of MAPK signaling pathways.     

Effects of shenmai injection on expression of TNF- α mRNA in peritoneal macrophages of scald mice

Objective To explore the effect of shenmai injection (SI) on expression of TNF- α mRNA in peritoneal macrophages (pMΦs) of scald mice. Methods BALB/c mice were inflicted with 11% of body surface area Ⅲ degree scald and injected intraperitoneally (ip) with SI daily for 5 days, and expression of TNF- α mRNA in pMΦs was determined by semi- quantitative RT- PCR.Results In scald mice, the expression of TNF- α mRNA in pMΦs increased significantly, but it was reduced obviously (P&lt;0.01) after SI administration, while the livability was increased markedly (P&lt;0.05). Conclusions For scald mice, the cause of death at early stage might be related to the high expression of TNF- α mRNA in pMΦs and the use of SI can decrease the death rate.     

Increase of TNFα-stimulated Osteoarthritic Chondrocytes Apoptosis and Decrease of Matrix Metalloproteinases 9 by NF-κB Inhibition

Objective To investigate the in vitro effect of caffeic acid phenethyl ester(CAPE),a NF-κB inhibitor,on the apoptosis of osteoarthritic(OA)chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2(MMP-2)and matrix metalloproteinase 9(MMP-9).Methods Annexin V-FITC/propidium iodide(PI)labeling and western blotting were used to observe and determine the apoptosis in TNFα-stimulated primary cultured osteoarthritic chondrocytes.Also,gelatin zymography was applied to examine MMP-2 and MMP-9 activities in supernatants.Results It was confirmed by both flow cytometry and western blotting that chondrocytes from OA patients have an apoptotic background.Use of CAPE in combination with 10 ng/mL of TNFα for 24 h facilitated the apoptosis.MMP-9 in the supernatant could be autoactivated(from proMMP-9 to active MMP-9),and the physiologic calcium concentration(2.5 mmol/L)could delay the autoactivation of MMP-9.The activities of MMP-2 and MMP-9 in the fresh supernatant increased significantly in response to stimulation by 10 ng/mL of TNFα for 24 h.The stimulatory effect of TNFα just on proMMP-9 was counteracted significantly by CAPE.Conclusion NF-κB could prevent chondrocytes apoptosis though its activation was attributed to the increase of proMMP-9 activity induced by TNFα(a pro-apoptotic factor).Therefore,therapeutic NF-κB inhibitor was a ’double-edged swords’ to the apoptosis of chondrocytes and the secretion of MMP-9.     

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis

Receptor activator of nuclear factor-kappaBligand(RANKL)promotes osteoclast formation,fusion,differentiation and activation.It plays akey role in osteoclast mediated bone erosion inrheumatoid arthritis(RA).Osteoprotegerin(OPG),as a soluble decoy receptor of RANKL,can prevent bone erosion in RA.Our previousstudies have shownthat Triptolide(TP),an activecompoundidentifiedin a traditional Chinese herb--Tripterygium wilfordii Hook F(TWHF),can ef-fectively inhibit bone destruction in ex…     

TRAF6截短体的构建及其对NF-κB信号通路的影响

目的 构建肿瘤坏死因子受体相关因子6(TRAF6)截短体,鉴定TRAF6截短体及对核因子-κB (NF-κB)信号通路的影响区域.方法 采用PCR技术扩增TRAF6 4个截短体(N1、N2、N3、C1)编码基因,分别将其构建在pFlag-CMV2重组载体,用荧光素酶报告基因方法对TRAF6 4个截短体功能活性进行验证....     

TRAF6在NF-κB信号通路中的修饰与降解

目的：研究肿瘤坏死因子受体相关因子6(TRAF6)在调节NF-κB通路中的作用机制。方法：利用转导了并稳定表达。TRAF6基因的293RZC细胞株，用荧光素酶报告基因系统测定CoA1对293RZC细胞NF-κB通路的激活，Westem Blot分析在NF-κB通路激活过程中TRAF6和IκBα的降解。结果：CoA1可诱导293RZC细胞NF-κB通路的激活，在这一过程中，TRAF6被修饰并降解，IκBα也发生降解，二者的降解被蛋白酶抑制剂MG132抑制。结论：TRAF6介导NF-κB通路的激活需要蛋白酶复合体的作用。     

Protective Effect of Sodium Tanshinone ⅡA Sulfonate on Injury of Small Intestine in Rats with Sepsis and Its Mechanism

Objective:To explore the protective effect of sodium tanshinoneⅡA sulfonate(STS) on small intestine injury in rats with sepsis and its possible mechanism.Methods:According to a random number table, 24 Tats were randomly divided into 3 groups:sham operation group(sham group),sepsis model group(model group) and STS treatment group(STS group),with 8 Tats in each group.A rat model of sepsis was induced by cecal ligation and puncture(CLP) for 5 h.STS(1 mg/kg) was slowly injected through the right external jugular vein after CLP.The histopathologic changes in the intestine tissue were observed under a light microscope,and the intestinal epithelial cell apoptosis was evaluated by terminal deoxynucleoddyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL) method.The expressions of Bcl-2,Bax and nuclear factorκB(NF-κB) p65 in the intestinal tissue was determined by Western blot.The levels of tumor necrosis factorα(TNF-α) and interleukin 6(IL-6) in the intestinal tissue were determined using enzyme-linked immuno-sorbent assay(ELISA). Results:Obvious injuries were observed in the intestinal tissue in the CLP group compared with the sham group. The expression of NF-κB p65 and the levels of TNF-αand IL-6 were up-regulated after CLP,the apoptosis of intestinal epithelial cells was increased after CLP,and the ratio of Bcl-2 to Bax was decreased.STS posttreatment could attenuate the injury on the intestinal tissue induced by CLP,decrease the apoptosis of intestinal epithelial cells and the levels of NF-κB p65,TNF-αand IL-6,and increase the ratio of Bcl-2 to Bax.Conclusion: STS can protect the small intestine in rats with sepsis,and the mechanism may be associated with the inhibition of intestinal epithelial apoptosis and the reduction of activation of inflammatory cytokines.     

Relationship between Single Nucleotide Polymorphism in TNF-α Gene Promoter Region and Inhibitory Effects of Triptolide on TNF-α Production in Peripheral Blood Mononuclear Cells of Healthy Humans

Tumor necrosis factorα(TNF-α)is ani mpor-tant cytokine that has extensive biological activity.It plays key rolesinthe pathogenesis of rheumatoidarthritis(RA).The TNF-αlocus has yielded a va-riety of polymorphic sites such as-308,-238, 498andso on[1].Amongthese polymorphic sites,ithas been demonstrated that TNF-α-308single nucle-otide polymorphism(SNP)is closely related withthe susceptibility,response to drugs and outcomeof RA[2-5].Triptolide that is a main active compo-nent of Tript…