Homocysteine-induced Enhanced Expression of Tissue Factor in Human Vascular Smooth Muscle Cells

The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hcy on the activity of nuclear factor-kappaB (NF-кB) and the expression of inducible nitric oxide synthase (iNOS) were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC (NF-кB inhibitor). Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.     

Expression of NF-κB in hRPE Cells Stimulated by PDTC and IL-1β

The constitutive expression of nuclear-factor-κB (NF-κB) in human pigment epithelial (hRPE) cells cultivated in vitro and the possible changes when incubated with PDTC and IL-I were investigated. The synchronized hRPE cells in vitro were divided into two groups. In nonPDTC group, hRPE cells were exposed respectively to IL-1β and NS (for detecting the constitutive expressions of NF-κB in hRPE cells) ; In PDTC group, PDTC-pretreated hRPE cells were exposed respectively to IL-1β?Aand NS. (for detecting the constitutive expression of NF-κB in PDTC-pretreated hRPE cells). The expression of NF-κB in hRPE cells in two groups was detected by immunofluorescence stain and flow cytometry. The results showed that the constitutive expression of NF-κB in hRPE cells in vitro was 8.05 %, and increased to 30.26 % by IL-1β. After PDTC pretreatment, the constitutive expression of NF-κB in hRPE cells was decreased to 3.74%, and 3.66 % by IL-l,respectively. It was concluded that the expressions of NF-κB in hRPE cells could be increased significantly by IL-1βand depressed effectively by PDTC. Also, PDTC could significantly inhibit the activation of NF-κB induced by IL-1β.     

Inhibition of Emodin on LPS-induced Nitric Oxide Generation by Suppressing PLC-γ Phosphorylation in Rat Peritoneal Macrophages

Objective To investigate the inhibitory mechanism of emodin on lipopolysaccharide (LPS)-induced nitric oxide (NO) generation in rat peritoneal macrophages. Methods NO production and iNOS expression were measured through nitrite assay and Western blotting assay, respectively. NF-κB activity and nuclei P65 expression were estimated by dual-luciferase and Western blotting assay, respectively. Intracellular free Ca2+ ([Ca2+]i) was detected using the ratiometric fluorescent calcium indicator dye, Fura-2, and a microspectrofluorometer. PLC-γ phosporylation was analyzed by Western blotting assay. Results First, emodin was found playing active roles in suppressing LPS-induced NF-κB activation in rat peritoneal macrophages. Second, emodin down-regulated transient [Ca2+]i and could increase in NF-κB upstream signal. Finally, emodin suppressed phosphorylation of PLC-γ by LPS stimulation in the upstream of [Ca2+]i. Conclusion Suppression of PLC-γ phosphorylation is involved in emodin inhibiting NO generation by LPS stimulation in rat peritoneal macrophages.     

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.     

穿透支原体脂质相关膜蛋白对小鼠巨噬细胞核因子κB活性的影响

目的研究穿透支原体脂质相关膜蛋白能否诱导激活小鼠巨噬细胞核因子κB（NF-κB）,以了解穿透支原体潜在的致病性。方法用从穿透支原体中提取的脂质相关膜蛋白刺激小鼠巨噬细胞,经Western blot和间接免疫荧光等方法检测NF-κB是否被激活,以及NF-κB抑制剂PDTC对NF-κB活性的影响。结果穿透支原体脂质相关膜蛋白能诱导小鼠巨噬细胞激活NF-κB,且NF-κB抑制荆PDTC能部分抑制穿透支原体脂质相关膜蛋白所诱导的NF-κB激活。结论穿透支原体脂质相关膜蛋白能激活小鼠巨噬细胞NF-κB,因而可能与其致病性有关。     

解脲脲原体LAMPs激活核因子κB诱导人单核细胞表达诱导型一氧化氮合酶和前炎症因子

目的分析解脲脲原体（Uu）的脂质相关膜蛋白（LAMPs）对人单核细胞（THP-1）表达诱导型一氧化氮合酶（iNOS）和前炎症因子白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）的影响。方法用Uu的LAMPs刺激人单核细胞后,Western blot法检测核因子κB（NF-κB）的激活和iNOS基因的表达,格氏试剂测定NO,ELISA法测定IL-1β、TNF-α和IL-6的含量。结果Uu LAMPs通过激活NF-κB诱导人单核细胞表达iNOS,且能以时间和剂量依赖方式刺激人单核细胞产生NO,诱导表达前炎症因子（IL-1β、TNF-α和IL-6）;NF-κB抑制剂PDTC可抑制NF-κB的激活、iNOS的表达及NO的产生。结论Uu LAMPs通过激活NF-κB途径诱导人单核细胞表达iNOS和前炎症因子,可能是产生某些疾病的一个重要的因素。     

调控高迁移组蛋白1诱导大鼠肺泡巨噬细胞表达诱生型一氧化氮合酶的信号通路

目的观察细胞外信号调节激酶(ERK)、p38分裂原激活的蛋白激酶(p38MAPK)及核因子-κB(NF-κB)在高迁移组蛋白1(HMGB1)诱导大鼠肺泡巨噬细胞表达诱生型一氧化氮合酶(iNOS)中的作用。方法体外培养的大鼠原代肺泡巨噬细胞(PRAMs)分别与ERK抑制剂PD98059、p38MAPK抑制剂SB203580和NF-κB抑制剂PDTC,以及PD98059联合SB203580共孵育2h后,培养基中分别加入重组人HMGB1作用6h。采用逆转录聚合酶链式反应检测培养细胞中iNOSmRNA表达,用Greiss法测定培养上清中NO2-/NO3-的含量。结果PD98059、SB203580和PDTC均可显著下调HMGB1诱导PRAMs表达iNOSmRNA和产生NO(P<0·05)。联合使用PD98059和SB203580可增强抑制iNOSmRNA表达和NO产生(P<0·05)。结论信号分子ERK、p38MAPK和NF-κB均参与了HMGB1诱导大鼠肺泡巨噬细胞表达iNOS和产生NO。     

血必净对LPS诱导血管内皮细胞NO产生和iNOS及核转录因子κB蛋白表达的影响

目的:探讨血必净对脂多糖( LPS )诱导血管内皮细胞（VEC）损伤的保护作用,研究在血必净干预下一氧化氮(NO)产生、诱导型一氧化氮合酶(iNOS)表达及信号转导机制。方法：将体外培养的VEC分为对照组、LPS(1 mg/L) 组、LPS(1 mg/L)+血必净( 25 g/L) 组、LPS( 1 mg/L )+吡咯烷二硫代氨基甲酸盐( PDTC, 20 μmol/L ) 组,在给予LPS前预先用血必净和PDTC孵育1 h。采用Western blotting法检测iNOS和核转录因子-κB p65 (NF-κB p65)蛋白的表达情况,采用Griess法检测上清液中NO的水平。结果：与对照组比较,LPS组VEC中NO水平、iNOS和NF-κB p65蛋白表达水平明显升高(P<0.01)。与LPS组比较,LPS+血必净组VEC中NO水平、iNOS和NF-κB p65蛋白表达水平明显降低(P<0.05或P<0.01);与LPS组比较,LPS+PDTC组VEC中NO水平和iNOS及NF-κB p65蛋白表达水平均明显降低(P<0.05或P<0.01)。LPS+血必净组与LPS+PDTC组比较,VEC中NO水平和iNOS蛋白表达水平差异无统计学意义(P>0.05),LPS+PDTC组VEC中NF-κB p65蛋白表达水平明显低于LPS+血必净组(P<0.05)。结论：血必净能抑制VEC中NO的产生和iNOS蛋白的表达,其机制可能是通过抑制NF-κB而抑制炎症反应。     

人胚胎干细胞源神经干细胞微囊泡对巨噬细胞极化的影响

目的: 探究人胚胎干细胞源神经干细胞微囊泡对巨噬细胞极化的影响。方法: Dil标记神经干细胞微囊泡,流式细胞仪和共聚焦显微镜观察THP-1巨噬细胞对微囊泡的内化;实时荧光定量PCR筛选微囊泡最适浓度;100 ng/mL 脂多糖刺激THP-1细胞6 h后,微囊泡处理12 h,实时荧光定量PCR检测巨噬细胞炎性因子IL-1β,肿瘤坏死因子α (tumor necrosis factor-α, TNF-α)以及IL-6的mRNA表达水平;微囊泡处理24 h, 蛋白免疫印迹法检测诱导型一氧化氮合酶 (inducible nitric oxide synthase, iNOS)和精氨酸酶1 (Arginase-1, Arg-1)蛋白表达水平;流式细胞术检测THP-1巨噬细胞M1极化表面标志CD86表达。结果： THP-1巨噬细胞在12 h和24 h均能明显内化神经干细胞微囊泡;0.12 μg/mL微囊泡具有最适调控作用;与未处理细胞相比,脂多糖处理后明显增加THP-1细胞IL-1β,TNF-α,IL-6等mRNA和iNOS蛋白,Arg-1蛋白表达差异无统计学意义(P<0.05);经神经干细胞微囊泡处理后THP-1细胞IL-1β,TNF-α,IL-6等mRNA和iNOS蛋白表达明显降低(P<0.05);与未处理细胞相比,脂多糖和微囊泡处理后THP-1巨噬细胞CD86表达差异无统计学意义。结论： 人胚胎干细胞源神经干细胞微囊泡能够减少THP-1细胞炎性细胞因子和iNOS表达,抑制巨噬细胞M1极化。     

M1型巨噬细胞相关因子在重度慢性牙周炎患者牙龈组织中的表达及其意义

目的：探讨M1型巨噬细胞相关因子诱导型一氧化氮合酶（iNOS）和信号传导及转录激活因子1（STAT1）在重度慢性牙周炎患者牙龈组织中的表达情况,阐明M1型巨噬细胞在慢性牙周炎发生发展中的可能作用。方法：选择15例重度慢性牙周炎患者的病损牙龈组织作为实验组,15例需要拔除第三磨牙患者的健康牙龈组织作为对照组。采用RT-PCR法检测2组患者牙龈组织中iNOS和STAT1 mRNA表达水平,Western blotting法检测2组患者牙龈组织中iNOS和STAT1蛋白表达水平。结果：与对照组比较,实验组患者牙龈组织中iNOS和STAT1 mRNA表达水平明显升高（P<0.01）;与对照组比较,实验组患者牙龈组织中iNOS和STAT1蛋白表达水平明显升高（P<0.01）。结论：在重度慢性牙周炎中M1型巨噬细胞介导的免疫应答增强,表明M1型巨噬细胞参与牙周组织的炎症反应和组织损伤。     

猪松弛素对人肺微血管内皮细胞产生一氧化氮的影响

目的 观察猪松弛素(pRLX)对人肺微血管内皮细胞(HMVECs)产生一氧化氮(NO)的影响,探讨这种效应的可能.机制.方法 Westemb lotting检测不同浓度pRLX作用下HMVEC s诱生型一氧化氮合成酶(iNOS)和原生型一氧化氮合成酶(cNOS)蛋白表达;Griess法检测不同浓度pRLX分别在不同的作用时间下对HMVECsNO产量的影响;Griess法检测选择性iNOS抑制剂氨基胍、非选择性NOS抑制剂L-单甲基精氨酸和NF-κB抑制剂吡咯烷二硫氨基甲酸对pRLX刺激HMVECs产生NO效应的影响.结果 pRLX促进HMVECs iNOS蛋白表达,但对cNOS表达影响不显著;DRLX呈浓度依赖性促进HMVECs NO产量,但24 h至96 h范围内各pRLX作用时间组之间NO产量无显著差异;氨基胍、L-单甲基精氨酸和吡咯烷二硫氨基甲酸均能阻断pRLX促进HMVECs产生NO的效应.结论 pRLX促进HMVECs iNOS表达和NO生成.     

核转录因子在肿瘤坏死因子-α所致内皮细胞凋亡中的作用

目的 探讨肿瘤坏死因子－α（ＴＮＦ－α）是否通过信号传导系统－核转录因子（ＮＦ－ｋＢ）的激活,导致诱导型一氧化氮合酶表达增加,引起内皮细胞凋亡。方法 体外培养牛肺动脉内皮细胞,观察ｉＮＯＳ抑制剂一氨基胍,以及ＮＦ－ｋＢ抑制剂－吡咯烷二硫氨基甲酸盐（ＰＤＴＣ）对ＴＮＦα所致内皮细胞凋亡的影响,观察细胞形态学变化,用ＤＮＡ电泳及ＤＮＡ断裂百分率确定细胞凋亡情况;Ｗｅｓｔｅｒｎｂｌｏｔ分析内皮细胞经ＴＮ