The Role of IκBα in TNF-α-induced Apoptosis in Hepatic Stellate Cell Line HSC-T6

To investigate the role of NF-κB in TNF-α induced apoptosis in HSC-T6, a mutant IκBα was transfected into HSC-T6 cells by lipofectin transfection technique and its transient effect was examined 48 h after the transfection. The activation of NF-κB was detected by immune fluorescence cytochemistry and Western blotting with anti-p65 antibody. The apoptosis and the rate of inhibition by TNF-α in both transfected and untransfected HSC-T6 cells were measured respectively by FAC-Scan side scatter analysis and MTF methods. Our results showed that TNF-α could activate NF-κB in untransfected cells but not in transfected HSC-T6 cells. The percentage of apoptosis in transfected cells were significantly higher than that in the untransfected ones （P〈0.01） and it was also true of the inhibition rate （P〈0.01）. It is concluded that the resistance of HSC-T6 towards apoptosis induced by TNF-α can be mediated by NF-κB activation. The inhibition of NF-κB activation by mutant IκBα can attenuate the resistance of HSC-T6 cells and increase its sensitivity to TNF-α.     

The Effects of PDTC plus Leflunomide and Cyclosporine on the NF-κB Signaling Pathway in Mouse-to-rat Cardiac Xenografts

In this study, the effects of pirrolidine dithiocarbamate （PDTC） plus leflunomide （Lef） and cyclosporine （CsA） on the NF-κB signaling pathway in mouse-to-rat cardiac xeno-transplantation models were investigated. NIH mice and Wistar rats served as donors and recipients respectively. Mouse-to-rat cardiac xenotransplantation was performed. The recipients were divided into 5 groups： group A （the control group）, group B （PDTC group）, group C （PDTC plus CsA group）, group D （PDTC plus Lef group） and group E （PDTC plus Lef and CsA group）. The expressions of IKKa/[3, NF-κB-P65, IκBct, ICAM-1 and NF-κB DNA binding activity in xenograft tissues were determined by immunohistochemistry and Western blot as well as electrophoretic mobility shift assay （EMSA）. The median survival time of cardiac xenografts in the control group, PDTC group, PDTC plus CsA group, PDTC plus Lef group and PDTC plus Lef and CsA group was （2.17±0.41）, （2.33±0.52）, （4.67±1.21）, （7.00±1.79） and （9.00±1.41） days respectively. The survival time of xenografts in the PDTC plus Lef and CsA group was significantly longer than that in other four groups （P〈0.05 for all）, that in the PDTC plus Lef group longer than that in the control group, PDTC group and PDTC plus CsA group （P〈0.05 for all）, that in PDTC plus CsA group longer than the control group and PDTC group （P〈0.05 for all）. The expressions of IKKα/β, NF-κB-P65, IκBα and ICAM-1 and NF-κ3 DNA binding activity were notably increased in mouse-to-rat cardiac xenografts. The expressions were decreased in the control group, PDTC group, PDTC plus CsA group, PDTC plus Lef and PDTC plus Lef and CsA group in turn. It was concluded that PDTC plus Lef and CsA can significantly suppress the expressions of IKKα/β, NF-κB-P65, IκBα, ICAM-1 and NF-κB DNA binding activity, thereby prolonging the survival of the cardiac xenografts.     

Increased expression and activity of MMP-9 in C-reactive protein- induced human THP-1 mononuclear cells is related to activation of nuclear factor kappa-B

The relation between the expression and activity of MMP-9 in C-reactive protein （CRP）-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B （NF-κB） was studied to investigate the possible role of CRP in plaque destabilization. Human THP-1 cells were incubated in the presence of CRP at 0 （control group）, 25, 50 and 100 μg/mL （CRP groups） for 24 h. In PDTC （a specific NF-κB inhibitor） group, the cells were pre-treated with PDTC at 10 μmol/L and then with 100 μg/mL CRP. The conditioned media （CM） and human THP-1 cells in different groups were harvested. MMP-9 expression in CM and human THP-1 cells was measured by ELISA and Western blotting. MMP-9 activity was assessed by fluorogenic substrates. The expression of NF-κB inhibitor α （IκB-α） and NF-κB p65 was detected by Western blotting and ELISA respectively. The results showed that CRP increased the expression and activity of MMP-9 in a dose-dependent manner in the human THP-1 cells. Western blotting revealed that IiB-α expression was decreased in the cells with the concentrations of CRP and ELISA demonstrated that NF-κB p65 expression in the CRP-induced cells was increased. After pre-treatment of the cells with PDTC at 10 μmol/L, the decrease in IκB-α expression and the increase in NF-κB p65 expression in the CRP-induced cells were inhibited, and the expression and activity of MMP-9 were lowered too. It is concluded that increased expression and activity of MMP-9 in CRP-induced human THP-1 cells may be associated with activation of NF-κB. Down-regulation of the expression and activity of MMP-9 may be a new treatment alternative for plaque stabilization by inhibiting the NF-κB activation.     

冬凌草甲素对多发性骨髓瘤细胞细胞周期和凋亡的影响及其机制研究

目的探讨冬凌草甲素(oridonin,Ori)对人多发性骨髓瘤RPMI 8226细胞的细胞增殖、细胞周期及凋亡的影响及其涉及机制。方法利用MTT比色法检测Ori对细胞增殖活性的影响;碘化丙啶(PI)单染流式细胞术检测细胞周期;TUNEL法检测细胞凋亡;Western blot技术检测p65核蛋白水平。结果 Ori能明显抑制RPMI 8226细胞增殖,其抑制作用呈时间、剂量依赖性;能同时引起细胞G2/M细胞周期阻滞、细胞凋亡和p65核蛋白水平下降;吡咯烷二硫代氨基甲酸盐(PDTC)抑制核因子κB(NF-κB)活性后进一步促进了Ori所致的细胞G2/M细胞周期阻滞和凋亡。结论 Ori能明显抑制RPMI 8226细胞增殖,引起G2/M细胞周期阻滞,诱导细胞凋亡,该效应与NF-κB活性抑制相关。     

Dual Effect of 3, 4-Dihydroxyacetophenone on LPS-induced Apoptosis in RAW264.7 Cells by Modulating the Production of TNF-α

To explore the pharmacological effect of 3,4-dihydroxyacetophenone (DHAP) on the apoptosis of RAW264. 7 macrophage cells and the mechanism, RAW264. 7 macrophage cells were treated with 100 or 500 mg/L lipopolysaccharide (LPS), with or without 10^-5 mol/L DHAP for 24 h. Trypan blue dye exclusion assay was used to assess cell viability. Cell apoptosis was morphological studied and flow cytometric assay was used. Tumor necrosis factor-α (TNF-α) level was measured by ELISA methods. IκB protein was determined by Western blotting. Our results showed that in 100 mg/L LPS stimulated macrophages, DHAP enhanced the cell apoptosis while in 500 mg/L LPS-stimulated macrophages, DHAP significantly inhibited the cell apoptosis. In both groups, DHAP increased the level of IκB but decreased the level of TNF-α. It is concluded that DHAP has dual effect on the apoptosis of RAW 264.7 cells treated with different concentrations of LPS. This effect may be due to the inhibition of activation of NF-κB and autocrine production of TNFα. Our study suggests that DHAP may have anti-inflammatory effect on LPS-activated macrophages.     

The elevated expression of osteopontin and NF-κB in human aortic aneurysms and its implication

The expression and significance of osteopontin (OPN) and NF-κB in patients with thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) were investigated.Thirteen TAA specimens,20 AAA specim...     

Effect of All-trans Retinoic Acid on Airway Inflammation in Asthmatic Rats and Its Mechanism

The inhibitive effects of all-trans retinoic acid (ARTA) on airway inflammation in asthmatic rats and its mechanism on the basis of the regulation of nuclear factor kappaB (NF-kB) were explored. Thirty-two SD rats were randomly divided into 4 groups: control group, asthma group,dexamethasone treatment group and retinotic acid treatment group. The total and differential cell counts in the collected bronchoalveolar lavage fluid (BALF) were measured. The pathological changes in lung tissues were estimated by scoring. The expression of NF-kB inhibitor (IkBa), NF-kB,intercellular adhering molecule-1 (ICAM-1) in lung tissue was detected by immunohistochemical method. The results showed that in the two treatment groups, the total cell counts and proportion of inflammatory cells in BALF were significantly reduced, but there was no significant difference in differential cell counts in BALF between them. The pathological changes in lung tissues in the treatment groups were significantly attenuated as compared with asthma group. Except the epithelial injury in retinotic acid treatment group was milder than in dexamethasone treatment group, the remaining lesions showed no significant difference between them. In the two treatment groups, the expression of IkBa was increased, while the expression of NF-kB and ICAM-1 decreased with the difference between the two groups being not significant. It was concluded that the similar anti-inflammatory effects and mechanism of ATRA on airway in asthmatic rats to those of dexamethasone were contributed to the increase of cytoplasmic IkBa content and suppression of NF-kB activationand expression.     

Expression of Nuclear Factor-κB in Mouse Uterus during Peri-implantation

To investigate the expression of the subunit p65 of NF-κB and inhibitor kappa B alpha (lκBα) in mouse uterus during peri-implantation, thereby investigating whether transient activation of nuclear factor-κB (NF κB) takes place during embryo implantation in mice. Immunohistochemical technique was used to examine the expression and localization of p65 in endometrium or deciduas, and Western blot analysis was employed to detect the levels of lκBα protein in mouse endometrium or deciduas. P65 protein was detected in stromal cells, epithelial cells of endometrium as well as in myometrium. Staining was predominately seen in the cytoplasm of the cells. Staining intensity for p65 was stronger in the epithelial compartment than the stromal compartment and myometrium. Staining intensity increased slightly during pregnancy, and it reached a high level on pregnancy day 5 and day 8. In contrast to p65, the level of IκBα protein was lowest on pregnancy day 5 in all groups. Our results suggested that NF κB may regulate embryo implantation by its transient activation in mice.     

Ursolic Acid Inhibits T-Cell Activation through Modulating Nuclear Factor-κB Signaling

<正>Objective:To investigate the effects of ursolic acid(UA) on T-cell proliferation and activation,as well as to examine its effect on nuclear factor-κB(NF-κB) signaling pathway in T cells.Methods:T-cells isolated from BALB/c mice were incubated with UA at concentrations ranging from 5-30μmol/L in the presence of phorbol 12-myristate 13-acetate(PMA) or PMA plus ionomycin.The proliferation of T cells was measured by the MTT assay.The expressions of CD69,CD25,and CD71 on T-cell surface were analyzed using flow cytometry. The level of interleukin-2(IL-2) in the culture supernatant of activated T cells was quantified by enzyme-linked immunosorbent assay(ELISA).The level of phosphorylated IκB-α(p-lκB-α) in total protein and p65,a subunit of NF-κB,nuclear translocation were measured by Western blot analysis.Results:UA in a dose-dependent manner significantly decreased the proliferation and inhibited the surface expressions of CD69,CD25,and CD71 in murine T lymphocytes upon in vitro activation(P0.01).Significant reduction of IL-2 production was found in activated T cells treated with UA(P0.01).The PMA-induced increase in p-lκB-αprotein was inhibited,and nuclear translocation of p65 from the cytoplasm was blocked by UA.Conclusion:UA is a potent inhibitor for T cell activation and proliferation;these effects are associated with the inhibition of NF-κB signaling pathway.     

Effect of Nuclear Factor-κB on Airway Remodeling in Asthmatic Rats

Summary In order to investigate the effect of nuclear factor-κB (NF-κB) on airway remodeling in asthmatic rats. 18 Wistar rats were divided into three groups: asthmatic group: pyrrolidine dithiocarbamate (PDTC) group, in which rats were injected intraperitoneally with NF-κB specific inhibitor PDTC (100 mg/kg) before ovalbumin (OVA) challenge; control group. The NF-κB activity and the expression of inhibitory protein κBα (I-κBα) in airway were detected by electrophoretic mobility shift assay (EMSA). Western blot and immunohistochemistry respectively. The infiltration of inflammatory cells, the number of Goblet cells, the area of collagen and smooth muscle in airway were measured by means of image analysis system. The results showed that with the up-regulation of airway NF-κB activity in asthmatic group, the number of goblet cells (3.08±0.86/100 μm basement membrane (BM)), the area of collagen (24.71±4.24 μm²/μm BM) and smooth muscle (13.81±2.11 μm²/μmBM) in airway were significantly increased (P<0.05) as compared with control group (0.14±0.05/100μmBM. 14.31±3.16 μm²/μm BM and 7.67±2.35 μm²/μm BM respectively) and PDTC group (0.33±0.14/100 μmBM, 18.16±2.85 μm²/μm BM and 8.95±2.16 μm²/μm BM respectively). However, there was no significant difference between PDTC group and control group (P>0.05). It was concluded that the activity of NF-κB is increased in airway of asthmatic rats. Inhibition of NF-κB, activation can attenuate constructional changes in asthma airway, suggesting NF-κB may contribute to asthmatic airway remodeling. XU Shuyun, female, born in 1970, M. D. Ph. D. This project was supported by a grant from Foundation for University Key Teacher by the Ministry of Education (2000 year).     

冬凌草甲素协同马法兰对人骨髓瘤RPMI-8226细胞增殖和凋亡的影响

目的: 观察冬凌草甲素（oridonin）和马法兰( melphalan)单药及联合用药对人骨髓瘤RPMI-8226细胞生长、凋亡和细胞周期的影响及二者的协同作用,阐明药物联合应用对骨髓瘤细胞的毒性增强作用。方法: 本实验共设4个RPMI-8226细胞处理组,即空白对照组（未加任何药物）、冬凌草甲素（10 μmol/L）组、马法兰（30 μmol/L）组和联合（冬凌草甲素 10 μmol/L + 马法兰 30 μmol/L）组。MTT法检测各用药组作用不同时间后RPMI-8226细胞的增殖抑制率,采用两药相互作用指数（CDI）评价联合用药性质;Annexin-Ⅴ/PI双染检测细胞凋亡率;流式细胞术检测药物作用前后细胞周期分布。结果:与空白对照组比较,冬凌草甲素组、马法兰组和联合组RPMI-8226细胞增殖抑制率明显增加(P<0.01),均具有时间-效应依赖性,联合组各时间点细胞增殖抑制率明显高于单药组(P<0.01)。各时间点CDI值均小于1,即冬凌草甲素可协同提高马法兰的细胞毒作用。冬凌草甲素和马法兰单独处理RPMI-8226细胞36 h,细胞凋亡率分别为15.01%±0.47％ 和20.03%±1.30％,而联合组细胞凋亡率为43.06%±1.96％,与单药组比较显著升高(P<0.01)。流式细胞术DNA含量分析,各药物组RPMI-8226细胞主要阻滞于G0/G1期,S期细胞比例明显减少,除冬凌草甲素组S期细胞比例外,其余药物组各期细胞比例与空白对照组比较差异均有统计学意义(P<0.05或P<0.01),联合组与单药组比较差异亦有统计学意义(P<0.01)。结论: 冬凌草甲素联合马法兰通过阻滞细胞周期和诱导细胞凋亡而抑制骨髓瘤细胞的增殖,其作用具有协同性。     

Protective effect and mechanism of sodium tanshinone II A sulfonate on microcirculatory disturbance of small intestine in rats with sepsis

To explore the protective effect of sodium tanshinone ⅡA sulfonate(STS) on microcirculatory disturbance of small intestine in rats with sepsis,and the possible mechanism,a rat model of sepsis was induced by cecal ligation and puncture(CLP).Rats were randomly divided into 3 groups:sham operated group(S),sepsis group(CLP) and STS treatment group(STS).STS(1 mg/kg) was slowly injected through the right external jugular vein after CLP.The histopathologic changes in the intestinal tissue and changes of mesenteric microcirculation were observed.The levels of tumor necrosis factor-α(TNF-α) in the intestinal tissue were determined by using enzyme-linked immunoabsorbent assay(ELISA).The expression of intercellular adhesion molecule-1(ICAM-1) in the intestinal tissue was detected by using immunohistochemisty and Western blot,that of nuclear factor κB(NF-κB) and tissue factor(TF) by using Western blot,and the levels of NF-κB mRNA expression by using RT-PCR respectively.The microcirculatory disturbance of the intestine was aggravated after CLP.The injury of the intestinal tissues was obviously aggravated in CLP group as compared with S group.The expression levels of NF-κB p65,ICAM-1,TF and TNF-α were upregulaed after CLP(P<0.01).STS post-treatment could ameliorate the microcirculatory disturbance,attenuate the injury of the intestinal tissues induced by CLP,and decrease the levels of NF-κB,ICAM-1,TF and TNF-α(P<0.01).It is suggested that STS can ameliorate the microcirculatory disturbance of the small intestine in rats with sepsis,and the mechanism may be associated with the inhibition of inflammatory responses and amelioration of coagulation abnormality.     

TNF-α Up-regulates Matrix Metalloproteinase-9 Expression and Activity in Alveolar Macrophages from Patients with Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality throughout the world. Unbalance of proteinases and an-tiproteinases is thought to be important in the pathogene-sis of COPD, as well as inflammation and oxidative stress. However, neutrophil elastase is likely to be the major proteinase involved in lung destruction in alpha-1 antitrypsin deficiency, and it may not be involved in COPD caused by inhalational exposures. Matrix metal-loproteinases…     

黄芩素抑制血管内皮生长因子表达及诱导多发性骨髓瘤细胞系RPMI-8226凋亡

目的:旨在探讨黄芩素(BAl)对血管生成的影响,及血管内皮生长因子(VEGF)对多发性骨髓瘤细胞系RPMI-8226细胞生长的影响.方法:体外培养RPMI-8226细胞系,不同浓度BAI处理此细胞系,用Annexin-V/PI双标流式细胞术检测BAI对RPMI-8226细胞系凋亡的作用,应用RT-PCR检测黄芩素对RP...     

Relationship between Carbachol Hyperstimulation-induced Pancreatic Intracelluar Trypsinogen and NF-κB Activation in Rats in vitro

The relationship between intracelluar trypsinogen activation and NF-κB activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist （carbachol） hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor （pefabloc） and NF-κB inhibitor （PDTC） in vitro. Intracelluar trypsin activity was measured by using a fluorogenic substrate. The activity of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-κB in pancreatic acinar cells treated with high concertrations of carbachol （10^-3 mol/L） in vitro was significantly decreased as compared with control group （P〈0.01 ）. The addition of 10^-2 mol/L PDTC resulted in a significant decrease of NF-κB activities in pancreatic acinar cells after treated with high concertrations of carbachol （10^-3 mol/L） in vitro, but the intracelluar trypsinogen activity was not obviously inhibited （P〉0.05）. It was concluded that intracelluar trypsinogen activation is likely involved in the regulation of high concertrations of carbachol-induced NF-κB activation in pancreatic acinar cells in vitro. NF-κB activation is likely not necessary for high concertrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.     

Growth inhibition and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780

Curcumin,aphenolicpigmentextractedfrom curcuma,isthemajoreffectivecomponentofthat traditionalChinesemedicineandusedasadietarycoloringagentandanaturalcondiment.Recentre searchreportshaveshownthatcurcumincouldse lectivelyinhibittheproliferationandinducetheap optosisofmanytumorcells,buttheinvolvedmech anismsstillunclear(1,2).Thephenomenonthatcurcu mininducesapoptosisoftumorcellscouldbeseen innumerouscancers,however,theeffectsofcurcu minonhumanovariancancercellshavenotbeenre portedyet.Inthisresear…