烧伤血清激活p38丝裂原活化蛋白激酶信号转导通路和核因子κB诱导血管内皮细胞血管细胞黏附分子1的表达

Activation of p38 MAPK signal transduction pathway by burn serum and the expression of VCAM-1 in HUVECs induced by NF-κB

目的观察烧伤血清对血管内皮细胞血管细胞黏附分子(VCAM)1表达的影响,探讨其信号转导机制。方法采用人脐静脉内皮细胞(HUVEC)培养模型,根据刺激物的不同分为正常血清组(正常血清刺激)、烧伤血清组(烧伤血清刺激)、SB203580组(烧伤血清+SB203580刺激)和吡咯烷二硫代氨基甲酸盐(PDTC)组(烧伤血清+PDTC刺激)。SB203580组和PDTC组在烧伤血清刺激前1h分别加入10μmol/L SB203580及10mmol/L PDTC.于烧伤血清刺激后即刻(0)、6、12、24和36h检测HUVEC内VCAM-1mRNA的转录水平。检测血清刺激24h后HUVEC膜表面VCAM-1表达水平、上清液中VCAM-1含量及HUVEC与外周血单核细胞(PBMC)之间的黏附情况。结果烧伤血清组刺激后VCAM-1mRNA的表达水平明显升高,在刺激24h时达高峰,随后下降。SB203580组及PDTC组刺激24h时HUVEC中VCAM-1mRNA表达明显下降,与正常血清组比较,差异无统计学意义(P>0.05).HUVEC膜表面VCAM-1表达水平:刺激24h时;烧伤血清组(66.5±6.2)显著高于正常血清组(19.1±1.9,P<0.05);而SB203580组及PDTC组,分别为21.7±2.3、23.1±2.4,与烧伤血清组比较明显降低(P<0.05),与正常血清组比较,差异无统计学意义(P>0·05).培养上清液中VCAM-1含量:刺激24h时,烧伤血清组(125±10)ng/L显著高于正常血清组(23±3)ng/L(P<0.05);而SB203580组(27±5)ng/L及PDTC组(29±5)ng/L均低于烧伤血清组(P<0.05).刺激24h时,烧伤血清组PBMC与HUVEC之间的黏附数为(197±11)%,较正常血清组(100±4)%显著增加(P<0·05),而SB203580组[(113±7)%]及PDTC组[(97±112)%]却明显低于烧伤血清组(P<0·05),与正常血清组比较,差异无统计学意义(P>0.05).结论烧伤血清可通过p38丝裂原活化蛋白激酶(MAPK)信号转导通路增强血管内皮细胞VCAM-1的表达,核因子(NF)κB系统参与了这一调控过程。

Objective To investigate the influence of burn serum on the expression of vascular cell adhesion molecule-1 （VCAM-1） of human umbilical vein endothelial cells（HUVECs） andits signal transduction mechanism. Methods HUYECs cultured in vitro were employed for the experiment, and were divided into normal control （NC, with addition of normal serum） , burn serum （BS, with addition of burn serum）, SB203580 （with addition of 10μmol/L SB203580 treatment 1 hour before burn serum treatment） and PDTC [ with 10mmol/L pyrrolidine dithiocarbamate （PDTC） 1 hour before burn serum treatment ] groups. Protein and mRNA expression of VCAM-1 in HUVECs was measured by flow cytometry and reverse transcription polymerase chain reaction （RT-PCR） respectively at O, 6, 12, 24 and 36 hours after burn serum treat-ment. The expression of VCAM-1 on HUVEC surface and the soluble VCAM-1 （ sVCAM-I ） content in HUVECs culture supernatants were measured by ELISA at 24 hours after the serum stimulation. Adherence of peripheral blood mononuclear leukocytes （PBMC） adherence to HUVECs was also observed in vitro. Resuits The expression of VCAM-1 mRNA increased obviously in BS group after the burn serum stimulation and reached peak level at 24 post stimulation hour（PSH） , and it decreased thereafter. The above expression was significantly decreased in SB203580 and PDTC groups at 24 PSH, but there was no difference compared with normal control （ P〉0.05 ）. The VCAM-1 expression on the membrane of HUVEC was evidently higher in BS group（66.5±6.2） than that in NC group（ 19.1±1.9, P 〈0.05）at 24 PSH, but it was decreased significantly in SB203580 （21.7±2.3） and PDTC （23.1±2.4） groups and there was no significant difference compared with NC group （ P〉0.05） , and which was evidently lower than that in BS group （ P 〈0. 05 ）. The VCAM-1 content in the supernatant of BS group（ 125±10 ng/L） was obviously higher than that in NC（23±3 ng/L）, SB203580（27±5 ng/L） and PDTC （29±5 ng/L）groups. （ P 〈0.05）. The number of PBMCs adherent to HUVECs in BS group[ （ 197±11 ） % ] was much larger than that in NC group[ （ 100±4 ）% I, SB203580 group[ （113±7）% ]or PDTC group[（ 97±112）% ] at 24 PSH （ P 〈0.05） , but no difference between NC group and SB203580 , PDTC groups （ P〉0. 05 ）. Conclusion Burn serum can enhance the expression of VCAM-1 in HUVECs through p38 MAPK signaling pathway, and the activation of NF-kB was also involved in this process.