葡萄糖对巨噬细胞不同脂蛋白受体表达的影响

目的研究不同浓度葡萄糖对巨噬细胞高密度脂蛋白受体(SR-BI)及凝集素样氧化型低密度脂蛋白受体(LOX-1)表达的影响。方法2003年5月至2005年1月在中国医科大学附属第一医院将5.6mmol/L、11.1mmol/L、16.7mmol/L和33.3mmol/L葡萄糖与诱导分化48h后的U937细胞共同孵育24h及16.7mmol/L葡萄糖与诱导分化48h后的U937细胞作用0,12,24,48,72h后,用Western印迹法检测SR-BI及LOX-1蛋白的表达。结果葡萄糖对巨噬细胞SR-BI蛋白表达量无显著影响(P>0.05)。葡萄糖浓度升高及作用时间延长可使LOX-1蛋白表达增加。结论葡萄糖诱导LOX-1蛋白表达增加并呈浓度和时间依赖性。     

糖基化终产物对U937巨噬细胞高密度脂蛋白受体蛋白表达的影响

目的　研究诱导分化及糖基化终产物 (AGEs)对人单核 /巨噬细胞 (U937细胞 )高密度脂蛋白受体 (SR BI)蛋白表达的影响 ,探讨AGEs和巨噬细胞SR BI在动脉粥样硬化中的作用。方法　U937细胞经PMA诱导分化 ,并将不同浓度或同一浓度AGEs与诱导分化 48h后的U937细胞共同孵育 ,用免疫细胞化学法和Western印迹法检测SR BI蛋白的表达。结果　诱导分化后U937细胞SR BI表达在 2 4、48h逐渐升高 ,72h下降 ;1 0 0、2 0 0和 40 0 μg/mlAGEs刺激后细胞表面SR BI蛋白表达量分别是BSA组的 1 44、2 38和 2 77倍 (P <0 0 5) ;40 0 μg/ml的AGEs作用 6、1 2、2 4、48h后 ,U937巨噬细胞SR BI蛋白表达量分别为 0h的 1 38、2 49、3 76和 4 2 5倍 (P <0 0 5)。结论　AGEs可增加U937巨噬细胞SR BI蛋白表达且呈浓度和时间依赖性。     

糖基化终产物对U937细胞巨噬细胞炎性蛋白-1α mRNA表达的影响

目的探讨糖基化终产物(AGEs)对U937细胞巨噬细胞炎性蛋白-1αmRNA表达的影响,及AGEs在糖尿病动脉粥样硬化中的作用。方法将U937细胞用不同浓度(100、200、400 mg/L)AGEs孵育24 h及同一浓度(400 mg/L)AGEs孵育0、12、24及36 h,采用原位杂交(PT-PCR)方法检测巨噬细胞炎性蛋白-1αmRNA的表达水平。结果对照组U937细胞内巨噬细胞炎性蛋白-1α呈弱表达;100、200及400 mg/LAGEs孵育24 h后,各组U937细胞内巨噬细胞炎性蛋白-1αmRNA表达的平均积分光密度值较对照组均显著升高(P<0.05);400 mg/L AGEs孵育12、24及36 h后,各组U937细胞内巨噬细胞炎性蛋白-1αmRNA表达的平均积分光密度值均高于0 h(P<0.05)。结论AGEs以时间及剂量依赖的方式促进U937细胞巨噬细胞炎性蛋白-1αmRNA的表达。     

葡萄糖和厄贝沙坦对单核巨噬细胞系凝集素样氧化型低密度脂蛋白受体表达的影响

目的研究不同浓度葡萄糖和厄贝沙坦对人单核巨噬细胞系(THP-1)凝集素样氧化型低密度脂蛋白受体mRNA及蛋白表达的影响,并探讨其可能的机制。方法 THP-1细胞经0.16μmol/L佛波酯诱导分化72 h后,将细胞分为对照组、不同浓度葡萄糖组(5、8、12和15 mmol/L)和厄贝沙坦干预组,葡萄糖组将不同浓度葡萄糖与诱导分化的巨噬细胞孵育24 h,厄贝沙坦干预组用厄贝沙坦预先孵育2 h后,再加入15 mmol/L葡萄糖共同孵育24 h。用荧光定量PCR和细胞酶联免疫法检测凝集素样氧化型低密度脂蛋白受体mRNA和蛋白的表达。结果与对照组相比,5 mmol/L葡萄糖组凝集素样氧化型低密度脂蛋白受体mRNA及蛋白表达差异无显著性(P>0.05),其余高糖组该受体mRNA和蛋白表达明显增加(P<0.05),并且其表达呈浓度依赖性;厄贝沙坦可显著抑制此作用,使该受体表达明显降低。结论高糖以浓度依赖方式上调凝集素样氧化型低密度脂蛋白受体mRNA和蛋白的表达,这可能是导致动脉粥样硬化发生的机制之一;厄贝沙坦可显著抑制这一作用,可能在抗动脉粥样硬化中起作用。     

C反应蛋白和高密度脂蛋白对血凝素样氧化型低密度脂蛋白受体1表达的影响

目的观察C反应蛋白和高密度脂蛋白对单核细胞株THP-1来源的巨噬细胞血凝素样氧化型低密度脂蛋白受体1蛋白和mRNA表达的影响,以及血凝素样氧化型低密度脂蛋白受体1表达的变化与细胞内胆固醇含量变化的关系。方法THP-1单核细胞株经佛波酯诱导分化为巨噬细胞。用不同浓度C反应蛋白或高密度脂蛋白在体外干预巨噬细胞,测定干预前后巨噬细胞血凝素样氧化型低密度脂蛋白受体1蛋白和mRNA表达的变化,并采用高效液相色谱测定巨噬细胞内胆固醇含量的变化。结果与对照组相比,C反应蛋白或高密度脂蛋白均可以诱导THP-1来源的巨噬细胞血凝素样氧化型低密度脂蛋白受体1蛋白和mRNA表达增加(P<0.05),C反应蛋白使巨噬细胞内总胆固醇和胆固醇酯含量显著增加(P<0.01),而高密度脂蛋白使细胞内总胆固醇和胆固醇酯显著降低(P<0.01)。结论C反应蛋白和高密度脂蛋白都能引起THP-1来源的巨噬细胞表面血凝素样氧化型低密度脂蛋白受体1表达上调,提示血凝素样氧化型低密度脂蛋白受体1并不是介导巨噬细胞参与炎症反应病理生理变化的关键性受体。     

氧化型低密度脂蛋白诱导U937细胞凋亡时细胞间粘附分子-1的表达时序

研究氧化型低密度脂蛋白诱导人单核细胞U937细胞吞噬脂质发生凋亡过程中对细胞间粘附分子－1表达的影响,用胸腺嘧啶核苷将U937细胞阻滞在G1期,流式细胞仪监测同步化处理效果;流式细胞术检测U937细胞泡沫化过程中细胞凋亡和细胞间粘附分子－1的表达;逆转录－聚合酶链反应分析细胞间粘附分子－1 mRNA的表达,结果显示,80mg/L氧化型低密度脂蛋白温育U937细胞48h可形成典型的泡沫细胞,72h可见凋亡细胞增多;氧化型低密度脂蛋白温育U937细胞12h即可检测到细胞间粘附分子－1表达,24h时达到最高值,48h略有降低,72h时则明显降低。结果表明,氧化型低密度脂蛋白可以促进U937细胞细胞间粘附分子－1表达,细胞间粘附分子－1的表达增强有利于巨噬细胞的趋化运动和对脂质的吞噬。     

糖基化终产物促进U937巨噬细胞高密度脂蛋白受体的表达

目的　研究糖基化终产物对U937巨噬细胞高密度脂蛋白受体表达的影响。方法　将糖基化终产物与诱导分化4 8h后的U937细胞共同孵育,用免疫细胞化学法和半定量逆转录聚合酶链反应检测细胞清道夫受体BI蛋白及mRNA的表达。结果　免疫细胞化学法检测10 0、2 0 0和4 0 0mg/L糖基化终产物刺激后U937巨噬细胞清道夫受体BI蛋白表达的平均积分光密度值分别为18.94±3.5 6、2 7.86±4 .39及35 .0 8±2 .37,较牛血清白蛋白组明显升高(13.76±3.74 ,P <0 .0 5 ) ;4 0 0mg/L糖基化终产物作用6、12、2 4及4 8h后,细胞清道夫受体BI表达的平均积分光密度值分别为16 .87±5 .6 5、2 5 .6 8±6 .97、35 .0 8±8.37及39.6 8±9.37,较0h组明显升高(12 .0 2±3.4 7,P <0 .0 5 )。半定量逆转录聚合酶链反应结果显示,4 0 0mg/L牛血清白蛋白及10 0、2 0 0和4 0 0mg/L糖基化终产物刺激后细胞清道夫受体BImRNA相对表达量分别是0 .32±0 .0 3、0 .5 3±0 .0 5、0 .6 4±0 .0 4和0 .89±0 .0 5 ;4 0 0mg/L糖基化终产物作用0、6、12、2 4及4 8h后,U937巨噬细胞清道夫受体BImRNA相对表达量分别为0 .4 1±0 .0 1、0 .6 2±0 .0 5、0 .80±0 .0 8、0 .87±0 .0 5、1.2 4±0 .13。结论　糖基化终产物可增加U937巨噬细胞高密度脂蛋白受体蛋白     

糖基化终产物对小鼠巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9活性的影响

目的 探讨糖基化终产物对小鼠巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9活性的影响.方法在培养的小鼠巨噬细胞株(J774A.1)中分别加入不同浓度(50、100、200及400 mg/L)的糖基化终产物干预24 h和同一浓度(200 mg/L)的糖基化终产物干预12、24及48 h,以无血清培养基和相应浓度的牛血清白蛋白为对照.用逆转录聚合酶链方法检测基质金属蛋白酶诱导物mRNA的表达,用酶联免疫吸附法检测上清中基质金属蛋白酶诱导物蛋白水平,用酶谱法检测上清中基质金属蛋白酶9的活性.结果 糖基化终产物干预组的基质金属蛋白酶诱导物mRNA表达水平和上清中蛋白水平与对照组比较差异有显著性,且随时间和浓度增加而增加(P<0.05).糖基化终产物干预组的上清中基质金属蛋白酶9的活性与对照组比较差异有显著性,且随时间和浓度增加而增加(P<0.05).结论 糖基化终产物促进小鼠巨噬细胞基质金属蛋白酶诱导物mRNA表达、蛋白分泌及基质金属蛋白酶9的活性;提示糖基化终产物可能通过调节巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9的活性而影响糖尿病动脉粥样硬化斑块的稳定性.     

凝集素样氧化型低密度脂蛋白受体1与糖尿病大血管病变

糖尿病患者倾向于早期发生广泛弥漫的动脉粥样硬化,而其确切机制尚未完全阐明.有研究提示低密度脂蛋白的氧化加速了糖尿病大血管病变的进程,凝集素样氧化型低密度脂蛋白受体1是一种新型氧化型低密度脂蛋白受体,糖尿病中血管内皮细胞凝集素样氧化型低密度脂蛋白受体1呈表达上调状态,而且可溶性凝集素样氧化型低密度脂蛋白受体1有可能成为血管性疾病的生物学指标.多项研究提示治疗2型糖尿病的相关药物可抑制凝集素样氧化型低密度脂蛋白受体1的表达.本文就凝集素样氧化型低密度脂蛋白受体1在糖尿病大血管病变中的作用、调节机制及干预措施作一综述.     

血凝素样氧化型低密度脂蛋白受体1基因沉默抑制人脐静脉内皮细胞表达分形趋化因子和单核细胞趋化蛋白1

目的探讨血凝素样氧化型低密度脂蛋白受体1基因沉默后能否抑制氧化型低密度脂蛋白诱导的分形趋化因子和单核细胞趋化蛋白1的表达。方法分别用不同浓度的氧化型低密度脂蛋白与人脐静脉内皮细胞共孵育,及预先对人脐静脉内皮细胞转染pGenesil-1 LOX-1 shRNA后再用氧化型低密度脂蛋白刺激,半定量RT-PCR、Western blot及酶联免疫吸附法检测血凝素样氧化型低密度脂蛋白受体1、分形趋化因子和单核细胞趋化蛋白1的mRNA和蛋白表达。结果氧化型低密度脂蛋白能呈浓度依赖性诱导血凝素样氧化型低密度脂蛋白受体1、分形趋化因子和单核细胞趋化蛋白1的mRNA和蛋白表达(P<0.01)。用RNA干扰抑制血凝素样氧化型低密度脂蛋白受体1的表达后,显著抑制了氧化型低密度脂蛋白诱导的分形趋化因子和单核细胞趋化蛋白1的mRNA和蛋白表达(P<0.01)。结论氧化型低密度脂蛋白能呈浓度依赖性诱导人脐静脉内皮细胞中分形趋化因子和单核细胞趋化蛋白1表达;这种诱导作用可以被血凝素样氧化型低密度脂蛋白受体1基因沉默抑制。     

Interleukin 18 stimulates release of soluble lectin-like oxidized LDL receptor-1 (sLOX-1)

Lectin-like oxidized LDL receptor-1 (LOX-1) appears to play crucial roles in atherosclerotic plaque rupture. We previously reported that circulating soluble LOX-1 (sLOX-1) levels are elevated in acute coronary syndrome (ACS) and that sLOX-1 can be a specific and sensitive biomarker for ACS. A proinflammatory cytokine interleukin 18 (IL-18) and its receptor are prominently expressed in atherosclerotic plaques. In addition, circulating IL-18 levels were reported to be high in ACS. In this study, we have examined if IL-18 can stimulate shedding of LOX-1 and subsequent release of sLOX-1. After transfection with LOX-1 cDNA, HEK-293T cells were incubated with or without IL-18. Cell-conditioned media and total cell lysates were subjected to immunoblot analyses with an anti-LOX-1 monoclonal antibody. In addition, ADAM10 cDNA, ADAM10 siRNA or control vector were also co-transfected into HEK-293T cells, and the cell-conditioned media and total cell lysates were subjected to LOX-1 immunoblotting after treatment with or without IL-18. The cell-conditioned medium/total cell lysate ratios in the amounts of LOX-1 or sLOX-1 were determined as sLOX-1 cleavage ratios. IL-18 (10-100ng/mL) stimulation increased the sLOX-1 cleavage by 3-4-fold in a concentration- and time-dependent manner. ADAM10 overexpression alone similarly enhanced the sLOX-1 cleavage. ADAM10 inhibition by ADAM10 siRNA transfection significantly suppressed IL-18-induced sLOX-1 cleavage. IL-18 similarly enhanced sLOX-1 cleavage in TNF-alpha-activated cultured endothelial cells, as well as LOX-1 transgenic mice in vivo. IL-18 appears one of the stimuli that enhance sLOX-1 release in ACS and ADAM10 may be involved in this process.     

LOX-1在氧化型低密度脂蛋白诱导U937细胞凋亡中的作用和卡托普利的干预作用

目的探讨ox-LDL受体LOX-1在ox-LDL诱导单核/巨噬细胞凋亡中的角色和卡托普利的干预作用。方法应用流式细胞术检测细胞凋亡,采用免疫组织化学技术和Western Blot测定Caspase3、8、9的表达。结果ox-LDL可诱导U937细胞的凋亡峰出现(12.773±1.413),应用卡托普利和LOX-1的阻断剂角叉菜胶、PIA后凋亡峰明显减少,其百分比分别为(1.02±0.166)(4.94±0.47)(2.62±0.656),同时U937细胞在用ox-LDL培养后代表线粒体通路的Cas-pase9,3和代表死亡受体通路caspases8,3表达都增加,应用卡托普利和LOX-1的阻断剂角叉菜胶、PIA后caspase3、8、9的表达均减少。结论ox-LDL是通过与其受体LOX-1结合发挥损伤作用的。卡托普利可以抑制ox-LDL对U937细胞凋亡的诱导作用,从而对单核巨噬细胞起到保护作用。     

Plasma sLOX-1 is a potent biomarker of clinical remission and disease activity in patients with seropositive RA

Objectives: Soluble lectin-like oxidized low-density lipoprotein receptor 1 (sLOX-1) is present in the circulation and synovial fluid in patients with rheumatoid arthritis (RA). The aim of this study was to assess whether sLOX-1 level is associated with clinical remission and disease activity in patients with RA.

Methods: Clinical and laboratory data were analyzed for 282 patients with RA. Plasma sLOX-1 level was measured by enzyme-linked immunosorbent assay (ELISA). The remission status and sLOX-1 levels were compared between four groups of patients based on the positivity of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs). Relationships between sLOX-1 level and the 28-joint Disease Activity Score with erythrocyte sedimentation rate (DAS28-ESR) were analyzed by multivariate logistic regression.

Results: The patients in the RF?+?ACPA?+?group tended to exhibit higher sLOX-1 levels when compared to the other three groups. In the RF?+?ACPA?+?group, the sLOX-1 level was significantly higher in the non-remission group than in the remission group, irrespective of treatment. Multivariate logistic regression showed significant correlations between sLOX-1 level and DAS28-ESR.

Conclusions: sLOX-1 level might be a useful biomarker for assessing clinical remission and disease activity in double-positive RA patients.     

Weight reduction can decrease circulating soluble lectin-like oxidized low-density lipoprotein receptor-1 levels in overweight middle-aged men

Circulating soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) has been reported to be associated with acute coronary syndrome, but its association with obesity has not been elucidated. In this study, we examined whether weight reduction would reduce the serum levels of sLOX-1 in a 12-week weight reduction intervention. Thirty-eight overweight middle-aged men were enrolled in the study, and 32 completed the intervention. The serum level of sLOX-1 was measured using a chemiluminescent enzyme-linked immunoassay. After the intervention program, body weight and the serum level of sLOX-1 decreased significantly (−7.5% ± 4.8% and −72.1% ± 35.9%, respectively). Changes in serum levels of sLOX-1 were positively correlated with changes in body weight (r = 0.54, P = .003), body mass index (r = 0.57, P = .001), body fat mass (r = 0.57, P = .002), total cholesterol (r = 0.41, P = .03), subcutaneous fat area (r = 0.50, P = .007), high-sensitivity C-reactive protein (r = 0.56, P = .002), leptin (r = 0.47, P = .01), and tumor necrosis factor-α (r = 0.32, P = .09); but no correlations were observed with fasting glycemic-related factors (blood glucose, hemoglobin A_1c, and insulin). Changes in body mass index and high-sensitivity C-reactive protein were selected as significant predictors of sLOX-1 changes by multiple regression analyses. These results suggest that LOX-1 induction may be related to adipocyte metabolism, inflammation, and immune response associated with obesity.     

Procoagulant activity of circulating microparticles is associated with the presence of moderate calcified plaque burden detected by multislice computed tomography

Background Circulating microparticles （MPs） have been reported to be associated with coronary artery disease （CAD）. In this study, we explored the relationship between MPs procoagulant activity and characteristics of atherosclerotic plaque detected by 64-slice computed tomography angiography （CTA）. Methods In 127 consecutive patients with CAD but without acute coronary syndrome and who under went 64-slice CTA, MPs procoagulant activity in plasma Coy a thrombin generation test）, soluble form of lectin-like oxidized low-density lipoprotein receptor-1 （sLOX-1） and N（epsilon）-（carboxymethyl） lysine （CML） circulating levels （by ELISA） were measured. A quantitative volumetric analysis of the lumen and plaque burden of the vessel wall （soft and calcific components）, for the three major coronary vessels, was performed. The patients were classified in three groups according to the presence of calcium volume： non-calcified plaque （NCP） group （calcium volume （%） = 0）, moderate calcified plaque （MCP） group （0 〈 calcium volume （%） 〈 1）, and calcified plaque （CP） group （calcium volume （%） 〉 1）. Results MPs procoagulant activity and CML levels were higher in MCP group than in CP or NCP group （P = 0.009 and P = 0.027, respectively）. MPs procoagulant activity was positively associated with CML （r = 0.317, P 〈 0.0001） and sLOX-1 levels （r = 0.216, P = 0.0025）. Conclusions MPs procoagulant activity was higher in the MCP patient group and correlated positively with sLOX-1 and CML levels, suggesting that it may characterize a state of blood vulnerability that may locally precipitate plaque instability and increase the risk of subsequent major cardiovascular events.     

Advanced Glycation End Products Stimulate Angiotensinogen Production in Renal Proximal Tubular Cells

Background

Elevated advanced glycation end products (AGE) in diabetes mellitus (DM) are implicated in the progression of DM-associated tissue injury, including diabetic nephropathy. The intrarenal renin-angiotensin system, in particular augmentation of angiotensinogen (AGT) in proximal tubular cells (PTC), plays a crucial role in the development of diabetic nephropathy. This study investigated hypothesis that AGE stimulates AGT production in PTC.

Ankylosing spondylitis macrophage production of higher levels of interleukin‐23 in response to lipopolysaccharide without induction of a significant unfolded protein response

Objective

Previous studies of the HLA–B27–transgenic rat model of ankylosing spondylitis (AS) suggested that macrophages develop an intracellular stress response called the unfolded protein response (UPR) and, as a result, secrete increased amounts of cytokines in response to Toll‐like receptor agonists such as lipopolysaccharide (LPS). Our objective was to determine whether macrophages from AS patients also undergo a UPR and secrete increased cytokines/chemokines in response to LPS.

Methods

Peripheral blood monocytes isolated from 10 AS patients and 10 healthy controls were differentiated in vitro with macrophage colony‐stimulating factor. Select samples were treated with interferon‐γ (IFNγ) to up‐regulate class I major histocompatibility complex (HLA–B) expression prior to stimulation with LPS for either 3 hours (for RNA) or 8–24 hours (for supernatant). UPR induction was assessed by measuring the expression of messenger RNA for ERdj4, BiP, and CCAAT/enhancer binding protein homologous protein 10 (CHOP).

Results

Although IFNγ treatment up‐regulated HLA–B expression (2‐fold; P < 0.0001), neither IFNγ nor LPS substantially enhanced BiP or CHOP expression (<1.3‐fold). ERdj4 expression increased weakly, but not significantly, in AS samples treated with IFNγ plus LPS (2.2‐fold; P = 0.31). In response to LPS, AS macrophages secreted more CXCL9, interleukin‐10 (IL‐10), IL‐12p70, IL‐23, and tumor necrosis factor α than did control macrophages (P ≤ 0.025). The most striking difference was observed for IL‐23 (median 265 pg/ml in AS patients versus 9 pg/ml in controls; P = 0.0007). We did not detect significant differences in IL‐6, IL‐8, or IFNβ production.

Conclusion

The greater production of IL‐23 by AS patient macrophages in response to LPS provides further support for the development of Th17/IL‐23–directed therapy. Since significant UPR induction was not detected in AS patient macrophages, the relationship between UPR and inflammatory cytokine production remains unclear.