百草枯处理HUVEC后NF-κB表达及其反义寡核苷酸干预作用

Expression of NF-κB and the influence of antisense oligonucleotide on NF-κB p65 in HUVEC treated with paraquate

编辑点评：百草枯（Paraquat，PQ）是一种速效触杀型灭生性除草剂，也是人类常见致命性的急性中毒源。肺是百草枯中毒的主要靶器官，特点是早期持续的肺泡炎和后期的肺纤维化病死率高，其发病机制不明，目前尚无特殊治疗。本期介绍四川大学华西医院何庆教授的课题组从发病机制，中西医结合治疗等方面所开展的系列研究。

Objective Activation of NF-κB（nuclear factor kappa B） can induce the synthesis of inflammatory mediators, and promote inflammatory reaction. The inflammatory reaction is involved in tissue injury in early phase of paraquat poisoning. We constructed a paraquat poisoning model by culturing human umbilical vascular endothelial cells （HUVEC） in vitro, to investigate the expression of NF-κB in paraquat poisoning and the influence of antisense oligonucleotide（ASON） on NF-κB, targeting its activity subunit p65. Methods A paraquat poisoning model was constructed by culturing human umbilical vascular endothelial cells （HUVEC） in vitro. The cell viability was detected at 48h by MTT test. We then calculated the half inhibiting concentration （IC50） of paraquat. The activation level and location of NF-κB p65 protein were detected by immunocytochemical method at 8 h, 24 h and 48h after treatment with 1.22 mM paraquat; ASON and sense oligonucleotide （SON） were transferred into HUVEC by liposome, then the levels of mRNA and NF-κB p65 protein were detected by RT-PCR and Western blot analysis respectively. HUVEC were divided into three groups, all treated with paraquat for 24h. ASON was transferred into the first group by liposome, SON was transferred into the second group by liposome too, the third group was not treated and serves as blank control; then we detected cell survival rate of every group by MTT test and statistically analyzed the differences among groups. Results After 48 h treatment with 11 serial concentrations of paraquat, cytotoxicity of this drug to HUVEC was assayed by the MTT, IC50=1.22 mM. Immunocytochemical analyses revealed that the activity subunit of NF-κB p65 was located in cytoplasm of normal HUVEC. When the cells were treated with paraquat for 8h, the staining density of cytoplasm increased and the nuclei showed positive staining. The number of positive nuclear staining cells increased at 24 h. Although most cells died at 48h, the nuclei of the survival cells were still positively stained. Both RT-PCR and Western blot analysis verified that p65 mRNA and protein were down-regulated after antisense oligo-nucleotide targeting p65 were transferred into HUVEC. The cell survival rates of the group treated with ASON were 96.1%, 86.2%, 67.6%, of the group treated with SON were 87.4%, 72.6%, 33.8%, and of the untreated group were 90.8%, 73.5%, 33.2%, at 24 h, 48 h, 72 h, respectively. The survival rates at 48 h and 72 h between ASON and SON group, ASON and untreated group were significant different. Conclusions NF-κB could be activated at the early phase of paraquat poisoning and be involved in inflammatory injury. The antisense oligo- nucleotide of NF-κB p65 subunit can inhibit the expression of NF-κB p65 protein, reduce the production of inflammatory mediators, lessen inflammatory reaction and inflammatory injury, thus increase the cell survival rate.