The Expression Of NF-κB And The Therapeutic Efficacy Of Antisense Oligonucleutide Against NF-κB P65 In HUVEC Treated With Paraquate

Objective: The activation of NF-κB(nuclear factor kappa B) can induce thesynthesis of inflammatory mediators, and promote inflammatory reaction.The inflammatory reaction has involved in tissue injury in early phase ofparaquat poisoning. So constructing a paraquat poisoning model withculturing human umbilical vascular endothelial cells (HUVEC) in vitro, toinvestigate the effects of NF-κB in paraquat poisoning and the therapeuticefficacy of antisense oligonucleotide(ASON) targeting its activity subunitp65.Methods: Constructing a paraquat poisoning model with HUVEC in vitro,detecting cell viability at 48h by MTT test, then calculating the half inhibitingconcentation (IC₅₀) of paraquat. Detecting the activation level and location ofNF-κB p65 protein by immuno-cytochemical method at 8h, 24h and 48hafter treatment with paraquat; synthesizing phosphorothioated ASON andsense oligonucleotide(SON), transferring them into HUVEC respectively by liposome, then detecting the level of mRNA and protein of NF-κB p65 byRT-PCR and Western blot analysis. HUVEC were devided into three groups,all group treated with paraquat for 24h, then, ASON was transferred into thefirst group by liposome, SON was transferred into the second group byliposome too, the third group were not treated as blank control; detecting cellsurvival rate of every group by MTT test. statistically analyzing thedifference among groups.Results: After 48h treated with 11 serial concentration of paraquat,cytotoxicity of this drug to HUVEC was assayed by the MTT, IC₅₀=1.22mM.Immunocytochemical analyses revealed that the activity subunit of NF-κBp65 was located in cytoplasm of normal HUVEC. When the cells weretreated with paraquat for 8h, the stain density of cytoplasm increased and thenuclear showed positive staining. The sum of positive nuclear got more at24h. Although most cells died at 48h, the nuclear of survival cells were alsopositive stained. Both RT-PCR and Western blot analysis verified the mRNAand protein of p65 were down-regulated after antisense oligo-nucleotidetargeting p65 were transferred into HUVEC. The cell survival rate of thegroup treated with ASON was respectively 96.1ï¼…, 86.2ï¼…, 67.6ï¼…at 24h, 48h,72h; correspondingly the group treated with SON was 87.4ï¼…, 72.6ï¼…, 33.8ï¼…;and the untreated group was 90.8ï¼…, 73.5ï¼…, 33.2ï¼…. The survival rate of 48hand 72h between ASON and SON group, ASON and untreated group showsignificant difference.Conclusion: NF-κB could be activated at the early phase of paraquat poisoning and involved in inflammatory injure. The antisense oligonucleotideof NF-κB p65 subunit can inhibit the expression of NF-κB p65 protein,reduce the production of inflammatory mediators, and lessen inflammatoryreaction and inflammatory injury, thus the cell survival rate could beincreased.