Recombinant adenovirus Ad-RUNrf2 reduces paraquat-induced A549 injury.
OBJECTIVE
An RU486-inducible recombinant adenovirus-Nrf2 construct (Ad-RUNrf2) was constructed and expressed in H460 cells to determine whether Nrf2 gene expression can be regulated and to observe the effect of the adenovirus Ad-RUNrf2 on inflammatory cytokines, oxidative stress and apoptotic factors that mediate paraquat (PQ)-induced A549 cell injury.


METHODS
The Nrf2 gene within the RU486 (mifepristone)-inducible system was introduced into an adenovirus vector. A549 cells were transfected with Ad-RUNrf2, and Nrf2 expression was detected using Western blotting and real time-polymerase chain reaction (RT-PCR). RT-PCR, Western blots and enzyme-linked immunosorbent assay were used for observing the effect of RU486-induced Nrf2 expression on the inflammatory cytokines (interleukin-6 (IL-6), IL-10 and tumor necrosis factor-α (TNF-α)), oxidative stress factors (catalase (CAT) and malondialdehyde (MDA)) and apoptosis factors (caspase-3, caspase-9 and cytochrome C) that mediated PQ-induced A549 cell injury.


RESULTS
After infection of H460 cells by Ad-RUNrf2, RT-PCR and Western blot analyses showed that Nrf2 expression increased with additional RU486 doses. IL-6 and TNF-α protein and gene expression levels were significantly reduced, and IL-10 protein levels were significantly increased. Although IL-10 expression increased, it remained significantly lower than that of noninduced adenovirus infection and the simple virus exposure group. RU486 induced a significant reduction in MDA expression and increased CAT protein levels. Caspase-9 and caspase-3 protein and gene expression levels decreased in the RU486 induction group (p < 0.05). Cytochrome C protein levels were not significantly reduced, but its gene expression was significantly decreased (p < 0.05).


CONCLUSIONS
Ad-RUNrf2 adenovirus was successfully constructed and can be stably expressed and regulated in cells. Ad-RUNrf2 can reduce PQ-induced inflammation, oxidative stress and apoptosis in A549 cells.
Emergency Department, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, China.
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Cai
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Lu
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Hong
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Jiang
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Wu
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Zheng
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Song
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Chang
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism; D005822:Genetic Vectors; D006540:Herbicides / Q000633:toxicity*; D006801:Humans; D051267:NF-E2-Related Factor 2 / Q000235:genetics* / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D010269:Paraquat / Q000633:toxicity*
D016207:Cytokines; D006540:Herbicides; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D010269:Paraquat