高糖诱导人脐静脉内皮细胞fractalkine表达及其凋亡的研究

目的探讨高糖诱导的人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVECs)fractalkine(FKN)表达与细胞凋亡的可能机制。方法对数生长期的HUVECs,分为正常组(N)、高糖组(HG)、氯化锂(lithium chloride,LiCl)组(LiCl)、高糖+LiCl组(HG+LiCl)。5.5 mmol/L的低糖DMEM培养的HUVECs种板10 h后按以上分组加入10 mmol/L的LiCl预处理2 h,再用33.3 mmol/L的高糖干预48 h后,用TUNEL法检测细胞凋亡,免疫组化SABC法检测β-catenin及p-GSK-3β(Ser9),免疫荧光FITC法检测FKN蛋白水平变化,RT-PCR检测GSK-3β和FKN mRNA水平变化。结果①与正常组比较,高糖诱导的HUVECs凋亡率明显增加(P<0.05)。与高糖组比较,LiCl可明显降低高糖环境下HUVECs的凋亡率(P<0.05),但仍高于正常组(P<0.05)。②与正常组比较,高糖组HUVECsβ-catenin及p-GSK-3β(Ser9)蛋白表达降低(P<0.05);GSK-3βmRNA水平及FKN蛋白和mRNA水平升高(P<0.05)。③与高糖组比较,LiCl+高糖组HUVECsβ-catenin及p-GSK-3β(Ser9)蛋白表达升高(P<0.05),GSK-3βmRNA水平(P<0.05)及FKN蛋白和mRNA(P<0.05)水平降低。结论高糖诱导的HUVECs凋亡可能与Wnt信号通路抑制和抑制FKN表达上调有关。     

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.     

Immunomodulation with Glatiramer Acetate Prevents Long-Term Inflammatory Pain

The potential application of glatiramer acetate (GA) therapy as a safe pharmacological treatment for the attenuation or prevention of long-term inflammatory pain in a rat model was explored. Peripheral inflammatory pain was induced by an injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paw. Genome-wide DNA microarray studies were used to survey molecular mechanisms involved in long-term GA analgesic responses. Administration of a single or double subcutaneous injection of GA before, or immediately after, intraplantar injection of pro-inflammatory CFA significantly attenuated allodynia and hyperalgesic pain responses up to ～3 weeks after CFA treatment. These beneficial effects of GA immunization therapy coincided with the attenuation of expression of the chemotactic fractalkine chemokine (CX3CL1) in the dorsal horn of the lumbar spinal cord (L4-L5) in response to CFA treatment, assessed by DNA microarray and confirmed immunocytochemically (ICC). This study is consistent with the hypothesis that a novel mechanism through which GA immunization therapy may beneficially influence long-term allodynia and hyperalgesia is through central regulation of fractalkine-mediated responses.     

CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway

The exact roles of activated microglia and fractalkine (CX3CL1)/fractalkine receptor (CX3CR1) signaling are not fully understood in brain ischemic injury and the findings reported are controversial. Here, we investigated the effects of CX3CR1 siRNA on the expression of CX3CR1, p38 mitogen-activated protein kinase (p38MAPK), Protein Kinase C (PKC) and inflammatory cytokines, microglia activation, white matter lesions, and cognitive function in mice treated with bilateral common carotid artery stenosis (BCAS) in vivo as well as effects of exogenous CX3CL1, CX3CR1 siRNA, and SB2035080 on expression of inflammatory cytokines in BV2 microglia treated with oxygen–glucose deprivation (OGD) in vitro. We showed that CX3CR1 siRNA significantly inhibited the increased expression of CX3CR1, p38MAPK, PKC as well as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and also attenuated microglia activation, white matter lesions, and cognitive deficits induced by BCAS in mice brain. We also showed that exogenous CX3CL1 could induce a further enhancement in TNF-α and IL-1β expression, which could be suppressed by CX3CR1 siRNA or by the p38MAPK inhibitor in OGD-treated BV2 microglial cells in vitro. Our findings indicated that CX3CL1/CX3CR1-mediated microglial activation plays a detrimental role in ischemic brain via p38MAPK/PKC signaling and also suggested that CX3CL1/CX3CR1 axis might be a putative therapeutic target to disrupt the cascade of deleterious events that lead to brain ischemic injury.     

Mito-KATP对缺氧复氧大鼠心肌微血管内皮细胞凋亡的影响及作用机制

目的探讨线粒体ATP敏感性钾通道(mito-KATP)开放影响缺氧复氧大鼠心肌微血管内皮细胞(MMECs)凋亡的作用机制。方法培养大鼠心肌微血管内皮细胞,随机分为四组:对照组(N组)、模型组(H/R组)、开放剂组(DZ组)、阻断剂组(5-HD组)。DZ组加入100μmol/L二氮嗪预处理2 h,5-HD组在加入100μmol/L二氮嗪前,先用100μmol/L 5-羟葵酸预处理2 h,然后上述两组和H/R组同样进行缺氧2 h复氧2 h。Hoechst染色方法观察凋亡细胞形态,Annexin V-FITC/PI双标记法测定各组细胞凋亡率、RT-PCR法检测各组NF-κB、FKN和p53 mRNA转录水平。结果与N组比较,H/R组可见大量细胞坏死、脱落,细胞凋亡率升高(P<0.01),NF-κB、FKN和p53 mRNA表达上调(P<0.01);与H/R组比较,DZ组可见部分细胞坏死脱落,细胞凋亡率降低(P<0.01),NF-κB、FKN和p53 mRNA表达下调(P<0.01);5-HD组与H/R组比较无显著差异。结论 mito-KATP开放可抑制缺氧复氧所致MMECs凋亡,其机制可能与抑制NF-κB、FKN及p53 mRNA表达有关。     

Microglia during development and aging

Microglia are critical nervous system-specific cells influencing brain development, maintenance of the neural environment, response to injury, and repair. They contribute to neuronal proliferation and differentiation, pruning of dying neurons, synaptic remodeling and clearance of debris and aberrant proteins. Colonization of the brain occurs during gestation with an expansion following birth with localization stimulated by programmed neuronal death, synaptic pruning, and axonal degeneration. Changes in microglia phenotype relate to cellular processes including specific neurotransmitter, pattern recognition, or immune-related receptor activation. Upon activation, microglia cells have the capacity to release a number of substances, e.g., cytokines, chemokines, nitric oxide, and reactive oxygen species, which could be detrimental or beneficial to the surrounding cells. With aging, microglia shift their morphology and may display diminished capacity for normal functions related to migration, clearance, and the ability to shift from a pro-inflammatory to an anti-inflammatory state to regulate injury and repair. This shift in microglia potentially contributes to increased susceptibility and neurodegeneration as a function of age. In the current review, information is provided on the colonization of the brain by microglia, the expression of various pattern recognition receptors to regulate migration and phagocytosis, and the shift in related functions that occur in normal aging.     

Fractalkine在糜烂-萎缩型及斑纹型OLP中表达的初步研究

目的：探讨fractalkine（FKN）在OLP中的表达及意义。方法：采用免疫组织化学方法,检测FKN在34例OLP中表达情况,分析FKN阳性着色与OLP临床类型、年龄、性别及发病部位的关系。结果：FKN在OLP角质形成细胞中高表达,FKN阳性细胞率为（45.4±16.2）%;在糜烂-萎缩型OLP中,角质形成细胞FKN染色分值（2.8±0.7）较斑纹型OLP染色分值（1.9±0.8）高,有显著性差异（P〈0.05）。OLP黏膜下层血管内皮细胞也发现FKN高表达。结论：FKN在角质形成细胞及血管内皮细胞中的高表达,可与T淋巴细胞相互作用,可能通过一种旁分泌反馈机制参与OLP发病机制,加重炎症反应。