In this research, we demonstrated for the first time that a low dose of plasmid DNA vector induces the production of low concentration of inflammatory cytokine IL-6 and TNF-α by in vitro LPS-stimulated macrophages. The novelty of this contribution lies in the fact that we evaluated this inflammatory suppression by a non-coding plasmid DNA in an endotoxemia model in rats and obtained a remarkable attenuation in the drop of MAP.
Since the sharp fall in MAP in case of endotoxemia can be a life-threatening situation, the maintenance of MAP is one of the major concerns in its treatment . Our data demonstrated that plasmid DNA, mainly at doses of 10 and 20 μg, when administered concomitantly with LPS in rats, significantly prevented the drop in MAP within the first hour of the experiment. This stabilization of MAP was maintained until the fourth hour after LPS injection. Although we cannot exclude that there may have been in vivo direct interaction between plasmid and LPS, this seems unlikely because this interaction should induce an inhibition of all parameters analyzed in a dose response manner. Our in vivo experiments demonstrated that 5 μg of plasmid was able to produce a decrease of the effect of LPS on the temperature of rats, but not on the ΔVHR and ΔVMAP, when compared with the doses of 10 and 20 μg. In contrast, 10 and 20 μg of plasmid diminished the effect of LPS on the ΔVHR and ΔVMAP, but not on temperature.
AVP and NO have been described as important mediators in septic shock [29–32]. AVP is a hormone that raises blood pressure by regulating vascular water balance and inducing vasoconstriction . The plasma concentration of AVP is increased in the first and second hour after administration of LPS in rats , as seen in our control 2 hours after LPS injection. More importantly, all tested doses of plasmid DNA were able to further increase the concentration of plasma AVP in LPS-injected rats at the second hour of experiment, in a dose dependent manner. Though plasmid DNA was responsible for the increase of AVP in the first 2 hours, its effect was indirect, since rats injected with only plasmid DNA did not have increased AVP concentrations. This increase in plasma AVP concentrations suggests it could be responsible for recovering MAP.
Concerning to NO, although some studies have demonstrated that specific iNOS inhibitors can reduce the MAP drop in animal models [34–36], this has not been observed in septic patients [37, 38]. Even so, we analyzed NO because it is considered one of the major factors responsible for refractory hypotension in sepsis, has its concentration increased after LPS injection , and appears to play a key inhibitory role in AVP released during endotoxemia, leading to hypotension . Apparently, NO over-production from endotoxin/cytokines-induced iNOS mediates a vasodilatation difficult to revert with vasoconstrictors . In our experiments, NO level was decreased until the fourth hour of the experiment only in the group administered with LPS and treated with 5 μg of plasmid DNA. Interestingly, 5 μg of plasmid DNA was not able to maintain the MAP in these rats. In contrast, 10 and 20 μg of plasmid DNA, which best stabilized the MAP in the second hour after LPS administration, were the doses that significantly increased the concentrations of NO when compared to rats injected with LPS only. Therefore, even with high production of NO, the LPS-injected rats treated with plasmid DNA did not show a drop in MAP.
Given that physiological changes in sepsis are in part caused by high concentrations of inflammatory mediators, it is reasonable to suppose that, among the treatments that have been investigated in sepsis, emphasis has been given to the inhibition of these mediators . In our experiment in vitro, plasmid DNA significantly decreased the production of IL-6 and TNF-α by LPS-stimulated macrophage cell line J774. The effect of plasmid DNA on LPS-stimulated J774 cells was more pronounced on the IL-6 than TNF-α, i.e., 99% reduction for IL-6 and 56% for TNF-α when compared with macrophages stimulated with LPS only. Similar results were obtained when our in vitro experiments were done with another plasmid (pVAX), suggesting that the anti-inflammatory property does not appear to be restricted to the pcDNA3 (data not shown). Because studies have indicated that Kupffer cells promote a significant role in the production of proinflammatory cytokines, such as TNF-α, IL-1 and IL-6, after stimulation with LPS [42–44], we evaluated IL-6 and TNF-α messages from liver. Interestingly, the results of real time RT-PCR showed that the in vivo gene expression of IL-6, but not TNF-α, was decreased significantly at 2 hours after plasmid DNA and LPS injection when compared with injection of LPS only, suggesting that IL-6 contributes to the stabilization of MAP. Such stabilization was not attributed to the TNF-α as it was decreased only at 4 hours after plasmid DNA and LPS injection. The role of IL-6 in murine sepsis has been somewhat controversial, since data from the literature are conflicting in relation to survival of IL-6 knockout mice subject to cecal ligation and puncture [45–47]. Nevertheless, IL-6 has pointed out as an important cytokine in sepsis with pro- and anti-inflammatory effects [48, 49], regardless of whether it is a disease biomarker or contributes to severity of sepsis . More importantly, IL-6 appears to be one of the best predictors to determine MODS, sepsis severity and mortality in both animal models [51, 52] and human [49, 53–56]. The former data from the literature supported our hypothesis that low expression of IL-6 induced by the plasmid DNA treatment was crucial to dampen the LPS-induced hypotension in rats.
AVP infusion has been suggested as an alternative therapy for septic shock patients that are refractory to usual vasopressor therapy . This therapy can be justified because plasma AVP concentrations in septic patients are maintained close to physiological level, although a lower blood pressure is noticeable . Because plasma concentrations of AVP might be inappropriately low in septic shock  and, as seen in this study, plasmid DNA can increase concentration of AVP and improve MAP, we suggested that plasmid DNA constitutes a novel approach with therapeutic potential in sepsis that may have clinical applications.