iNOS-targeted 10-23 DNAzyme reduces LPS-induced systemic inflammation and mortality in mice

Sepsis and/or systemic inflammatory response syndrome are leading causes of death in intensive care unit patients. NO is a critical player in the pathogenesis of bacterial sepsis. Several studies demonstrate elevation of iNOS in LPS-induced acute inflammatory responses and mortality; however, the effectiveness of its therapeutic suppression in systemic inflammation is largely controversial. Earlier, we have reported that DNAzymes specific to iNOS mRNA efficiently suppress iNOS expression in LPS-stimulated J774 murine macrophages. In the present study, we explored the effects of two of these DNAzymes in BALB/c mice model of LPS-induced lethal systemic inflammation. Experimental animal groups receiving previous injections of iNOS-specific DNAzyme (100 microg, i.p.) showed significantly reduced mortality. Total cell counts of peritoneal lavage and histopathological studies of tissues demonstrated substantial reduction in the leukocytic infiltration and edema in DNAzyme-treated mice. In addition, DNAzyme-injected animals displayed significantly decreased IL-12 serum level, whereas the levels of IL-1[beta], IFN-[gamma], and TNF-[alpha] also declined to a great extent. DNAzyme treatment resulted in significantly reduced NO levels in serum and peritoneal lavage, confirming functional suppression of iNOS gene in LPS-injected mice. These DNAzymes were also able to limit excessive NO production by cytokine and LPS co-challenges in cultured peritoneal macrophages from DNAzyme-treated mice. Estimation of iNOS mRNA and protein expression in the peritoneal macrophages of DNAzyme-administered animals further confirmed the iNOS gene knockdown. All these results indicated that iNOS-specific DNAzymes reduce inflammatory responses and enhance survival in murine model of LPS-induced lethal systemic inflammation.