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Narsing Rao, Sindhu Saraswathy; In the retinal mitochondrial oxidative stress NLRC4 inflammasome silencing attenuates photoreceptor pyroptosis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1806.
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© ARVO (1962-2015); The Authors (2016-present)
We recently developed retinal iNOS over expressing transgenic animals with phenotype of photoreceptor mitochondrial oxidative stress without inflammatory cell infiltration. In these iNOS +/+ mice, we found upregulation of NLRC4 and robust activation of caspase 1, IL-1B and IL-18, the key molecules of inflammasome complex. The aim of the present study is to prevent the oxidative stress mediated retinal pyroptosis by silencing NLRC4-inflammasome activation in the transgenic mice.
A groups of iNOS +/+ mice were intravitreally injected with NLRC4 siRNA and other group of mice were similarly injected with scrambled RNA sequence (controls). A third group of WT mice injected without such injections and scrambled RNA were also used as controls. The retinas were dissected out 3 days after the injection and subjected to detect inflammasome complex genes and its signaling molecules by qPCR array (SABioscience). Localization of NLRC4 and activated caspase1 was performed by immunohistochemistry. The retinas from these groups of mice were also subjected to qPCR to detect the levels of pyroptosis and apoptosis genes.
: In the retina of animals injected with the siRNA, NLRC4 and the inflammasome molecules were significantly (P<0.05) down-regulated compared to the iNOS+/+ mice injected with the scrambled RNA. NLRC4 was localized in the retina of iNOS+/+ animals specifically to the photoreceptors and outer plexiform layer and it was also co-localized with activated caspase 1. In contrast, in the WT control mice NLRC4 and activated caspase 1 was undetectable. There was down-regulation of pyroptosis related genes in the NLRC4 siRNA treated mice whereas in the iNOS+/+ mice pyroptosis associated genes were up-regulated. The apoptosis related key molecule caspase 3 was not detected in the retina of all 3 groups of animals.
Silencing of NLRC4 prevented the activation of inflammasome complex, in particular caspase 1, IL-18 and IL-1B. The expression of NLRC4 was primarily localized to the retinal photoreceptors at the sites of retinal mt.oxidative stress. These results reveal that NLRC4 plays a pivotal role in induction of photoreceptor pyroptosis in the retinal mt.oxidative stress and siRNA directed at the NLRC4 with local intraocular delivery could prevent oxidative stress mediated pyroptotic cell death.
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