| Background:NLRP6 is part of the leucine-rich repeat-containing receptors(NLR)innate immune receptor group of intra-cytoplasmic pattern recognition receptors,which forms a protein complex known as the “inflammasome” typically leading to cytosolic pattern recognition receptor(PRR)and apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC).Further connected with caspase-1 it cleaves onto their biologically active forms,the precursors of IL-1? and IL-18,NLRP3 as well NLRC4.Like other members of the NLR family,NLRP6 can be activated by exogenous or endogenous signaling to form inflammasome.This encourages IL-1? and IL-18 to mature by triggering caspase-1 and subsequent inflammatory reactions.Recently,the pro-inflammatory effect of NLRP6 inflammasome in gingival fibroblasts and acute liver injury was elucidated.NLRP6 is the first member known to inhibit innate immune response-related signaling pathways.There are some reports on the antiinflammatory role of NLRP6 independent of inflammasome activation in colitis,intestinal injury,and transmissible enteritis.Yet,the role of NLRP6 in the brain is still unclear and its effect on I/R injury has not been reported.Objective:To explore the role of NLRP6 in I/R injury and further clarifies the effects of NLRP6-independent inflammasome activation and NLRP6-dependent inflammasome.Methods:(1)Used a middle cerebral artery occlusion/reperfusion model(MCAO)to imitate ischemic injury by SD rats and divided the rats at random into 6 groups: sham,6h,12 h,24h,48 h,72h MCAO.These rats were anesthetized with 4% chloral hydrate(1ml/100g)by intraperitoneal injection.Then these animals were applied a nylon filament which featured a rounded tip to the interior of the left middle cerebral artery for 1h.The issues which were used for detecting the peak of NLRP6 through related experiments-Western Blot and RT-qPCR were applied common kit to gather all protein from the same part of the cerebral cortex.(2)Divided the rats at random into 6 groups: MCAO,MCAO + control siRNA,and MCAO + NLRP6 siRNA(NLRP6 209/864/1439/2519),The left ventricle was injected before setting the MCAO model and western blot was used to screen the most interfering small molecule interference fragments(siRNA)of NLRP6.(3)Adopted 2,3,5-triphenyltetrazolium chloride(TTC)staining to assess cerebral infarction volume in sham/MCAO/MCAO+ control siRNA/ MCAO+ NLRP6 siRNA groups.Similarly,assessed all neurological function scores and measured the brain water content after 48 h of reperfusion.HE and Nissl's staining were used to observe the histological changes.MPO staining was applied to explore in the experience.(4)Used the Western blot to detect the expression of NLRP6?IL-1?,IL-18,cleaved Caspase-1 and the activity of IL-1? and IL-18 was assessed by ELISA in sham/MCAO/MCAO+ control siRNA/ MCAO+ NLRP6 siRNA groups.(5)In sham/MCAO/MCAO+ control siRNA/ MCAO+ NLRP6 siRNA groups,CO-IP was used to observe the NLRP6-ASC binding state.Results:(1)The mRNA level of NLRP6 expression peaked at 24 h of reperfusion and NLRP6 expression peaked at 48 h of reperfusion.(2)In four NLRP6 siRNA,the interference effect of NLRP6-rat-209 is most obvious.(3)NLRP6 siRNA decreased cerebral I/R injury and enhanced neurological function.(4)NLRP6 siRNA decreased the expression and activity of IL-1?,IL-18,and cleaved caspase-1.(5)NLRP6 siRNA decreased the NLRP6-ASC binding state.Conclusions:(1)NLRP6 expression peaked at 48 h of reperfusion and NLRP6 expression peaked at 48 h of reperfusion.(2)NLRP6 has a proinflammatory effect by dependent inflammasome in cerebral ischemia-reperfusion injury in rats. |
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