补体C1q肿瘤坏死因子相关蛋白1通过激活p38信号通路增加内皮细胞黏附
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(河南省人民医院 阜外华中心血管病医院,河南省郑州市 450000)

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孟华,硕士,主治医师,研究方向为冠状动脉粥样硬化发病机制,E-mail为menghua0706@126.com。通信作者陈岩,主任医师,硕士研究生导师,研究方向为冠心病的诊断及治疗,E-mail为doctorchen04@163.com。

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河南省科技厅基础与前沿技术研究计划项目(142300410264)


CTRP1 promotes the adhesion of human umbilical vein endothelial cells through activation of the p38 signaling pathway
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Henan Provincial People's Hospital, Fuwai Central China Cardiovascular Hospital, Zhengzhou, Henan 450000, China)

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    摘要:

    目的 探讨补体C1q肿瘤坏死因子相关蛋白1(CTRP1)对人脐静脉内皮细胞(HUVEC)黏附分子表达及黏附功能的影响及其分子机制。方法 体外实验采用培养HUVEC和人单核细胞白血病细胞株THP-1细胞。(1)采用不同浓度rCTRP1(10、100、1 000 μg/L)刺激HUVEC 24 h,用实时荧光定量PCR和Western blot分别检测血管细胞黏附分子1(VCAM-1)和细胞间黏附分子1(ICAM-1)mRNA和蛋白表达水平;(2)不同浓度rCTRP1(10、100、1 000 μg/L)或TNF-α(10 μg/L)分别刺激HUVEC 24 h,通过共孵育HUVEC和THP-1 在倒置荧光显微镜下观察单个核细胞-内皮细胞黏附情况;(3)对照组:生理盐水和BSA(10 μg)为阴性对照,TNF-α(3 μg)为阳性对照;实验组:rCTRP1(3、10 μg)分别腹腔注射小鼠,倒置显微镜下实时观察小鼠肠系膜上动脉中白细胞的滚动及黏附情况;(4)1 000 μg/L rCTRP1刺激HUVEC(15、30、60 min),采用Western blot检测p38、ERK、JNK、p65的磷酸化水平,应用SB203580抑制p38MAPK信号通路后,Western blot检测黏附分子VCAM-1、ICAM-1的蛋白表达。结果 (1)rCTRP1刺激HUVEC后,黏附分子VCAM-1和ICAM-1的mRNA和蛋白表达水平显著升高(P<0.05),呈剂量依赖性,尤以ICAM-1明显;(2)体外实验:随着CTRP1刺激剂量增加,单个核细胞-内皮细胞黏附数显著增加,差异有统计学意义(P<0.05);体内实验:小鼠腹腔注射rCTRP1后,血管内白细胞滚动速度显著下降,黏附在内皮的细胞数目显著增加,差异均有统计学意义(P<0.05);(3)rCTRP1刺激HUVEC后p38和p65磷酸化水平显著增加,ERK、JNK磷酸化水平无明显变化,SB203580抑制p38MAPK信号通路后,rCTRP1诱导的VCAM-1和ICAM-1蛋白表达显著下降(P<0.05)。结论 CTRP1诱导内皮细胞分泌VCAM-1和ICAM-1,增加内皮细胞黏附能力,其机制可能是通过激活p38MAPK信号通路。

    Abstract:

    Aim To investigate the effects of C1q/TNF-related protein 1 (CTRP1) on the expression of adhesion molecules and adhesion function of human umbilical vein endothelial cells(HUVEC) and related mechanisms.Methods HUVEC were cultured and treated with different doses of recombinant CTRP1(0,0,1 000 μg/L). After 24 hours of stimulation, the gene expression and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were detected by Real-time fluorescence quantitative PCR and Western blot respectively. Fluorescence labeled THP-1 monocytes were incubated with HUVEC which were pre-treated with recombinant CTRP1(0,0,1 000 μg/L)or TNF-α(10 μg/L)for 24 h. The number of adhesion monocytes was counted by fluorescent microscope. In in vivo adhesion experiment, recombinant CTRP1 (3 or 10 μg), positive control factor TNF-α (3 μg), or negative control protein bovine serum albumin (BSA) was injected intraperitoneally for 2 h. Real-time monitoring microscopy was used to examine the rolling and adhesion of monocytes to the wall of mesenteric arteries. HUVEC were incubated with recombinant CTRP1(1 000 μg/L) and the expression levels of phospho-p38MAPK,phospho-ERK,phospho-JNK and phospho-p65 were detected by Western blot. Simultaneously, HUVEC pre-treated with SB203580 (the p38MAPK special inhibitor) were incubated with recombinant CTRP1(1 000 μg/L). The expression of adhesion molecules were detected by Western blot. Results After 24 h stimulation with recombinant CTRP1, protein and mRNA levels of adhesion molecules(VCAM-1 and ICAM-1) were significantly increased in a concentration-dependent manner in HUVEC(P<0.05). CTRP1 dose-dependently induced the adhesion of THP-1 cells to HUVEC in vitro and interactions of leukocytes to the wall of mesenteric arteries in vivo. CTRP1 activated the p38 mitogenprotein kinase(MAPK) pathway with concurrent phosphorylation of the p65 in HUVECs. Consistently administration of p38 inhibitor SB203580,virtually abolished CTRP1-induced expression of VCAM-1 and ICAM-1. Conclusion CTRP1 can induce the expression of VCAM-1 and ICAM-1 in HUVEC and promote the adhesion function of HUVEC through the activation of p38 MAPK signaling pathway.

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孟华,安松涛,张燕,陈岩.补体C1q肿瘤坏死因子相关蛋白1通过激活p38信号通路增加内皮细胞黏附[J].中国动脉硬化杂志,2019,27(1):34~39.

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  • 收稿日期:2018-05-18
  • 最后修改日期:2018-09-02
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  • 在线发布日期: 2019-01-21