26S蛋白酶体LMP2亚基参与调节血管内皮细胞对氧化应激的耐受性形成

Involvement of 26S proteasome LMP2 subunit in development of tolerance against oxidative stress in vascular endothelial cells

目的:观察在氧化应激耐受及氧化应激损伤的血管内皮细胞(VECs)26S蛋白酶体LMP2亚基的表达变化情况,并评价26S蛋白酶体LMP2亚基在调节VECs对氧化应激耐受性中的可能作用。方法:建立H2O2诱导氧化应激损伤及氧化应激预处理VECs模型;采用细胞免疫荧光标记联合Western blotting法检测26S蛋白酶体LMP2的表达;采用脂质体LipofectamineTM2000转染LMP2反义/正义寡核苷酸;检测培养上清中乳酸脱氢酶(LDH)的活性及丙二醛(MDA)的含量。结果:H2O2可剂量及时间依赖性地诱导VECs氧化应激损伤,培养上清中MDA浓度及LDH活性明显增加;H2O2(10μmol/L,24 h)预处理可诱导26S蛋白酶体LMP2亚基的高表达及细胞对H2O2(500μmol/L,3 h)诱导氧化应激的耐受,培养上清中MDA浓度及LDH活性明显减少;与H2O2(500μmol/L,3 h)处理组比较,采用干扰素γ(IFN-γ,20μg/L,48 h)可诱导VECs 26S蛋白酶体LMP2亚基高表达,也可诱导VECs对H2O2的耐受,其培养上清中MDA浓度及LDH活性明显降低,而转染LMP2-反义寡核苷酸可抑制IFN-γ诱导的培养上皮细胞26S蛋白酶体LMP2亚基高表达并阻断细胞对H2O2(500μmol/L,3 h)的耐受。结论:26S蛋白酶体LMP2亚基参与VECs对氧化应激耐受性的形成。

AIM： To observe the expression of 26S proteasome LMP2 subunit in vascular endothelial cells（VECs） under oxidative stress,and to evaluate its role in the development of tolerance against oxidative stress in VECs.METHODS： The cell model of H2O2 preconditioning-induced oxidative tolerance was established in VECs.The expression of LMP2 was detected by cellular immunofluorescent labeling and Western blotting.The LMP2 anti-sense and sense oligonucleotides were transfected into VECs by LipofectamineTM 2000.The damages of VECs were evaluated by detecting the activity of lactate dehydrogenase（LDH） and the concentration of malondialdehyde（MDA） in the culture medium.RESULTS： H2O2（500 μmol/L for 3 h） induced oxidative stress in VECss in a dose-and the activity of time-dependent manner,characterized by the increase in the concentration of MDA and LDH in the culture medium.Pretreatment with H2O2（10 μmol/L for 24 h） up-regulated the expression of LMP2.Meanwhile,the capacity of cellular tolerance against oxidative stress induced by H2O2 was increased as the concentration of MDA and the activity of LDH in the culture medium significantly decreased.Compared with H2O2 group,up-regulation of LMP2 by IFN-γ pretreatment（20 μg/L for 48 h） increased the tolerance of VECs against H2O2 injury,and the MDA conentration and the activity of LDH in the culture medium also significantly decreased.Transfection with LMP2 antisense oligonucleotide partly inhibited the increased expression of LMP2 induced by IFN-γ in VECs and abolished the tolerance against H2O2.CONCLUSION： The 26S proteasome LMP2 subunit is associated with the development of the tolerance against H2O2-induced oxidative stress in VECs.