杀菌性/通透性增加蛋白模拟肽对内毒素致血管内皮细胞损伤的保护作用

Protective effects of bactericidal/permeability increasing protein simulated peptide on human umbilical vascular endothelial cells injured by lipopolysaccharides

目的 通过杀菌性 通透性增加蛋白 (BPI)模拟肽 (BNEP)拮抗内毒素 (LPS) ,观察对血管内皮细胞分泌白细胞介素 - 6 (IL - 6 )、肿瘤坏死因子 -α(TNF -α)以及表达细胞间黏附分子 -1(ICAM - 1)的影响 ,探讨BNEP对LPS致血管内皮细胞损伤的保护作用。 方法 采用体外培养的原代人脐静脉内皮细胞 (HUVEC)为模型 ,分别观察单纯应用LPS、BNEP以及不同剂量BNEP与LPS作用后各细胞因子的分泌表达。IL - 6、TNF -α应用双抗夹心酶联免疫吸附法 (ELISA)测定 ;ICAM - 1采用生物素 -抗生物素 -过氧化物酶复合物 (SABC) -cy3免疫荧光染色 ,激光共聚焦扫描显微镜下观察其表达情况。 结果 LPS 10ng ml可使IL - 6分泌量显著增加 ;BNEP 6 0 μg ml即可显著降低 10ng mlLPS诱导的IL - 6分泌 ,随BNEP浓度增加IL - 6分泌量呈降低趋势 ,达2 4 0 μg ml时 ,IL - 6分泌量与单纯应用BNEP组比较 ,差异无显著性意义 (P >0 .0 5 )。BNEP 12 0 μg ml与LPS 10ng ml作用后 ,可使TNF -α分泌量降低 94 % ;BNEP 2 4 0 μg ml可完全阻断TNF -α的分泌。激光共聚焦扫描显微镜观察结果显示 ,BNEP与LPS作用后 ,ICAM - 1在细胞膜及细胞浆的表达明显减弱。 结论 BNEP在阻断LPS对细胞的激活和损伤。

Objective To investigate the effects of bactericidal/permeability increasing protein (BPI) simulated peptide (bactericidal neutralizing endotoxin protein, BNEP) and lipopolysaccharides (LPS) on secretions of IL 6, TNF α and ICAM 1 of primarily cultured human umbilical vascular endothelial cells (HUVEC) and to explore the protective role of BNEP on HUVEC injured by LPS. Methods Primarily cultured HUVEC was used as model. We observed IL 6,TNF α secretions and ICAM 1 expression stimulated respectively by LPS (E.Coli.O111:B4), BNEP alone or different dosages of BNEP preincubated with LPS before being added to the cells in tissue culture plates. Supernatants were collected and the cytokines detected using method of enzyme linked immunosorbent assay (ELISA). The expression of ICAM 1 was explored and stained by CY 3 using confocal laser scanning fluorescence microscope. Results The findings demonstrated that IL 6 and TNF α dramatically increased under stimulation by LPS (E.Coli.O111:B4) in a dose of 10 ng/ml. However, the combination of LPS with BNEP could decrease the Interleukin 6 secretion. BNEP (60 μg/ml) preincubated with LPS 10 ng/ml for 30 minutes could effectively block the increase of IL 6. TNF α decreased 94% using BNEP 120 μg/ml compared with LPS 10 ng/ml alone. The results of confocal laser scanning fluorescence microscope showed that BNEP could also significantly decrease the expression of ICAM 1 stimulated by LPS. Conclusions These results suggest that BNEP, as a simulated peptide of BPI, can effectively block the increases of IL 6,TNF α and ICAM 1 secretions of HUVEC and has a strongly protective effect on the injury of LPS. It implies that BNEP can be a promising agent for prevention and treatment of systemic inflammatory response syndrome (SIRS) and sepsis.