百草枯中毒早期大鼠肾组织中不规则趋化因子的表达

目的通过观察不规则趋化因子(Fractalkine,FKN,又称CX3CLl)在百草枯(Paraquat,PQ)中毒早期肾组织中的表达,探讨FKN在PQ致大鼠急性肾损伤中的作用。方法 SD大鼠80只,按随机数字法分为染毒组(40只)和对照组(40只)。染毒组腹腔注入PQ(22 mg/kg),对照组给予等量生理盐水。每组分别在染毒后0.5、1、3、5 d处死大鼠。测定血清肌酐及尿素氮,HE染色观察肾组织病理改变,免疫组化法检测FKN的表达。结果 PQ染毒后大鼠出现肾功能损害,血肌酐及尿素氮同步升高,且随染毒时间延长,肾功能损害逐渐加重,与同时间对照组相比,差异有统计学意义(P<0.05)。镜下观察,染毒组大鼠肾组织病理改变为肾小球体积增大,肾小管上皮细胞肿胀及管腔变窄;此后随观察时间延长,可见水肿渗出、坏死,间质炎性细胞浸润增加,肾小管内蛋白管型。免疫组化发现:染毒组在染毒后0.5、1、3、5 d,FKN表达呈逐渐增高趋势(P<0.05),且明显高于对照组,差异具有统计学意义(P<0.01)。结论 FKN在PQ早期大鼠肾组织中可表达,并与急性肾损伤的发病过程紧密相关。     

CX3CR1 receptor is up-regulated in monocytes of coronary artery diseased patients: impact of pre-inflammatory stimuli and renin-angiotensin system modulators

Fractalkine/CX3CR1 pathway is considered a major modulator of atherosclerosis. In the present study, expression of CX3CR1 on PBMCs/monocytes of healthy individuals and coronary artery diseased patients was initially assessed by flow cytometry. Effects of pre-inflammatory cytokines interferon (INF)-gamma and tumor necrosis factor (TNF)-alpha on expression of CX3CR1 and a single representative of each major chemokine family (CCR5 and CXCR4) were further assessed in three cell models: THP-1 monocytes, Jurkat T lymphocytes and primary monocytes isolated from healthy donors. Finally, effects of angiotensin-converting enzyme (ACE) inhibitors captopril, lisinopril and angiotensin receptor blocker (ARB) losartan on chemokine receptor expression were evaluated in the same cell models either in a naive or stimulated state. INF-gamma significantly affected the chemokine receptor phenotype of THP-1 cells by increasing the rate of CX3CR1-positive cells. Pre-treatment with the ACE inhibitors, captopril and lisinopril, and the ARB, losartan, did not influence these effects. Captopril and lisinopril similarly had no effect on either stimulated or naive primary monocytes. Yet, a small but repeatable increase in CX3CR1 expression after treatment with losartan was noted. Nevertheless, the latter observation did not retain statistical significance after applying the Bonferroni correction. In conclusion, our data did not indicate any significant effect of the ACE inhibitors on the chemokine receptor phenotype of monocytes.     

FKN对人外周血单个核细胞PKC-ERKl/2活化和TNF-α表达的影响

目的 探索FKN-CX3CR1在单个核细胞中可能存在的信号传导途径及促进动脉粥样硬化形成的机制,并探讨蛋白激酶C（PKC）在其中作用。方法 （1）用Ficoll密度梯度离心法分离抗凝人外周血单个核细胞。（2）将每份提取的单个核细胞分为4组：对照组、FKN组、Ro31-8220（PKC特异性阻断剂）和PD98059（ERK1/2特异性阻断剂）组。（3）用Western blot法检测单个核细胞中磷酸化ERK1/2表达。（4）用ELISA法检测培养液中TNF-α的表达。结果 （1）FKN组磷酸化的ERK1/2和TNF－α表达较对照组显著增多（P<0.05）。（2）Ro31-8220组磷酸化的ERK1/2和TNF－α表达较FKN组显著减少（P<0.05）。结论 FKN－CX3CR1可能通过PKC/ERK途径诱导单个核细胞TNF-α的表达,最终促进动脉粥样硬化的形成和进展。     

FKN对外周血单个核细胞ERK1/2活化和TNF-α表达的影响及ERK1/2的临床作用

目的 探讨趋化因子(FKN)对单个核细胞细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)1/2和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达的影响及ERK1/2在其中的作用.方法 抗凝血用Ficoll密度梯度离心法分离外周血单个核细胞.将每份提取的单个核细胞分为3组:空白对照组、FKN组、FKN+PD98059(ERK特异性阻断剂)组;分别应用Western印迹法和酶联免疫法检测各组单个核细胞中磷酸化ERK1/2及TNF-α的表达水平.结果 FKN诱导组磷酸化的ERK1/2和TNF-α表达较空白组显著增多(P<0.05);PD98059组磷酸化的ERK1/2和TNF-α表达较FKN组显著减少(P<0.05).结论 FKN-CX3CR1可能通过增加单个核细胞磷酸化ERK1/2和TNF-α的表达而促进动脉粥样硬化的进展,FKN可能是通过ERK1/2途径诱导单个核细胞TNF-α的表达.     

Enhanced recruitment of CX3CR1+ T cells by mucosal endothelial cell-derived fractalkine in inflammatory bowel disease

BACKGROUND & AIMS: Fractalkine (FKN/CX3CL1) is a unique chemokine combining adhesive and chemotactic properties. We investigated FKN production by the mucosal microvasculature in inflammatory bowel disease (IBD), its capacity for leukocyte recruitment into the gut, and the number of CX3CR1+ cells in the circulation and mucosa of IBD patients. METHODS: The expression of FKN by human intestinal microvascular endothelial cells (HIMECs) and CX3CR1 by circulating cells was evaluated by flow cytometry, and mucosal CX3CR1+ cells were enumerated by immunohistochemistry. The capacity of FKN to mediate leukocyte binding to HIMECs was assessed by immunoblockade, and to induce HIMEC transmigration by a Transwell system. RESULTS: The spontaneously low HIMEC FKN expression was enhanced markedly by tumor necrosis factor-alpha plus interferon-gamma stimulation, or direct leukocyte contact. This effect was significantly stronger in IBD than control HIMECs. Up-regulation of HIMEC FKN expression was dependent on p38 and extracellular signal-regulated kinase phosphorylation, as was abrogated by selective mitogen-activated protein kinase inhibitors. Circulating T cells contained significantly higher numbers of CX3CR1+ cells in active IBD than inactive IBD or healthy subjects, and IBD mucosa contained significantly more CX3CR1+ cells than control mucosa. Antibody-blocking experiments showed that FKN was a major contributor to T- and monocytic-cell adhesion to HIMECs. Finally, FKN enhanced the expression of active beta1 integrin on leukocytes and mediated leukocyte HIMEC transmigration. CONCLUSIONS: In view of the capacity of FKN to mediate leukocyte adhesion, chemoattraction, and transmigration, its increased production by mucosal microvascular cells and increased numbers of circulating and mucosal CX3CR1+ cells in IBD point to a significant role of FKN in disease pathogenesis.     

趋化因子FKN对单个核细胞TNF-α和MMP-2表达的影响及ERK在其中的作用

目的探讨趋化因子Fmetalkine（FKN）对单个核细胞肿瘤坏死因子-α（Tumor Necrosis Factor,TNF-α）和基质金属蛋白酶-2（matrix metal proteinase-2, MMP-2）表达的影响及细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）在其中的作用。方法抗凝血用聚蔗糖-泛影葡胺密度梯度离心法分离人外周血单个核细胞;将提取的单个核细胞分为：空白对照组、FKN组及PD98059（ERK阻断剂）组;分别应用ELISA和明胶酶谱法检测各组单个核细胞中TNF-α和MMP-2表达情况。结果FKN诱导组TNF-α表达较空白组明显增多（P〈0．05）,FKN诱导组MMP-2表达较空白组明显减少（P〈0．05）;PD98059组TNF-α和MMP-2表达均较FKN组明显减少（P〈0．05）。结论趋化因子FKN可以诱导单个核细胞TNF-α的表达而促进动脉粥样硬化的形成和进展,这一作用可能是通过ERK途径实现的;FKN可以抑制单个核细胞MMP-2的表达,而这一作用可能与ERK无关。     

进展性脑梗死患者血清HMGB1、FKN动态变化研究

目的探讨高迁移率族蛋白B1(HMGB1)及不规则趋化因子(FKN)在进展性脑梗死中的可能作用机制。方法用酶联免疫吸附试验(ELISA)法检测急性脑梗死患者不同时期血清HMGB1及FKN水平变化,根据斯堪的纳维亚脑卒中量表(scandinavian stroke scale,SSS)评分分为进展组和非进展组。根据梗死体积将180例患者分为大梗死灶组、中梗死灶组、小梗死灶组。另纳入30例健康体检者作为对照组。结果 180例急性脑梗死患者中有35例(19.44%)发展为进展性脑梗死。进展组SSS基线评分低于非进展组,差异有统计学意义(P0.05)。进展性组患者发病后1 d、7 d、14 d血清HMGB1、FKN水平高于非进展组和对照组,有统计学意义(P0.05)。梗死灶体积大则血清HMGB1、FKN水平高(P0.05)。结论血清HMGB1、FKN增加可能通过增加炎症反应促进动脉粥样硬化形成和进展,加重脑组织损伤,导致脑梗死患者病情进展。     

Multiple labeling of a potent CX3CR1 antagonist for the treatment of multiple sclerosis

Several methods for the preparation of five isotopologues of the CX₃CR1 antagonist 1 were developed. Volatile and radioactive 1‐chloro‐ and 1‐bromo‐ethyl‐benzene was handled in [2′‐¹⁴C] and [3′, 5′‐³H] labeling of 1. d ‐Leucinol ((R)‐2‐amino‐4‐methylpentan‐1‐ol) was labeled as [1‐¹⁴C] and [4‐¹⁴C] via a Wittig reaction using Garner's aldehyde and a Strecker amino acid synthesis with d ‐acylase resolvation, respectively. A [²H₁₀]d ‐leucinol was used for the stable labeled [M + 10] isotopologue. The products were isolated with 97.6–100% stereo chemical and radiochemical purity as for specific activity 768 GBq/mmol and 1.6–2.0 GBq/mmol, respectively.