Nrf2 Inhibits NLRP3 Inflammasome Activation Through Regulating Trx1/TXNIP Complex In Cerebral Ischemia Reperfusion Injury

Background: NLRP3?NOD-like receptor pyrin domain containing 3?is a member of NOD-like receptor family of cytoplasmic recognition receptors.NLRP3 can develop self-oligomerization under stress and apoptosis-related microparticle proteins ASC?apoptosis-associated speck-like protein containing?can help raise pro-caspase1 to form NLRP3 inflammatory complex.Recent studies have shown that the activation of NLRP3 inflammasome can promote post-cerebral ischemia-reperfusion inflammatory cascade to exacerbate cerebral ischemia-reperfusion injury,but so far has not been able to find an effective negative regulator for the process of NLRP3 inflammasome activation,therefore,it is important to find a negative regulator that inhibit the activation of NLRP3 inflammatory complex and to elucidate its regulation.Objective: To investigate whether Nrf2 can regulate the activation of NLRP3 inflammasome by regulating Trx1 / TXNIP complex in rat cerebral ischemia-reperfusion injury and elucidate its mechanism.Method:?1?Using Longa suture method to establish Middle Cerebral Artery Occlusion?MCAO?model for SD rats.The protein and mRNA levels of NLRP3 expression were observed at 4h,8h,12 h,24h and 48 h after cerebral ischemia-reperfusion injury.The rats were perfused with normal saline and decapitated after anesthetization,then the tissues were extracted and used for Western blot and Real-time qPCR analysis for the time point selection of highest expression of NLRP3 inflammasome.?2?siRNAs of NLRP3?NLRP3₁980,NLRP3₂185,NLRP3₂262?and the control siRNA used in the control group were built.The siRNA was injected intraperitoneally 24 hours before the establishment of MCAO model.Western blot and RT-qPCR were used to test the interference effect and selection of the most effective siRNA.?3?The expression of NRLP3 inflammasome,caspase-1,IL-1? and IL-18 were detected by Western blot and RT-qPRC in Sham,MCAO,MCAO + control siRNA and MCAO + NLRP3 siRNA groups.The neurological score of each group was observed,the volume of cerebral infarction was measured by TTC staining,and the protein concentration of IL-18 and IL-1? was detected by ELISA.?4?The Trx1 siRNA was used to knockdown Trx1,and the knockdown effect was tested by RT-qPCR and Western Blot.Expression of Trx1,NLRP3 inflammasome,Cytoplasm and Nucleus TXNIP were detected by Western blot in Sham,MCAO,MCAO + control siRNA and MCAO + Trx1 siRNA groups.?5?Nrf2 siRNA was used for Nrf2 knockdown and tBHQ was used for Nrf2 activation,tBHQ was dissolved in dimethylsulfoxide?DMSO?,and the intraperitoneally injection was divided into three times of injections at intervals of 8h before MCAO model.The Nrf2 knockdown and excitatory efficiency was detected by Western blot and RT-qPCR.The expression of Nrf2,NLRP3 inflammasome,Caspase-1,IL-18 and IL-1? were detected by Western blot in Sham,MCAO,MCAO + DMSO,MCAO + tBHQ,MCAO + control siRNA groups,and the protein concentration of IL-18 and IL-1? was detected by ELISA.?6?Establish the MCAO model of Trx1 knockdown by siRNA and Nrf2 activated by tBHQ.The expression of TXNIP in the cytoplasm and nucleus were examined by Western blot analysis.Result:?1?The expression of NLRP3 inflammatory complex reached its peak at 24 hours after I / R injury,and then decreased at 48 hours.?2?Among those NLRP3 siRNAs,NLRP3₂185 interference effect is the most obvious,up to 65%.?3?Compared with MCAO group,MCAO + control siRNA group showed no interference effect,and compared with MCAO+ control siRNA group,MCAO + NLRP3 siRNA group showed that NLRP3,Caspase-1,downstream inflammatory factors IL-18 and IL-1? expression were significantly reduced,TTC staining showed that cerebral infarction volume was significantly reduced,and behavioral defects also improved.?4?Compared with MCAO + control siRNA group,MCAO + Trx1 siRNA group showed that the expression of NLRP3 inflammasome was increased,and the nuclear TXNIP expression was decreased,cytoplasm TXNIP expression was increased.?5?There was no significant difference in the expression of Nrf2 between MCAO + DMSO group and MCAO group.Compared with MCAO + DMSO group,the expression of Nrf2 in MCAO + tBHQ group was significantly higher,and the expression of NLRP3 inflammasome,cytoplasm TXNIP,Caspase-1,IL-18 and IL-1? were significantly decreased.Compared with MCAO + control siRNA group,MCAO + Nrf2 siRNA group showed that the expression of NLRP3 inflammasome,cytoplasm TXNIP,Caspase-1,IL-18 and IL-1? were significantly increased and the expression of nuclear TXNIP was decreased.In addition,compared with MCAO + tBHQ group,MCAO + tBHQ + Trx1 siRNA group showed that the expression of TXNIP in the cytoplasm was significantly increased,and the expression of TXNIP in the nuclear was significantly decreased.Conclusion:?1?NLRP3 inflammasome reached the highest level 24 hours after I / R injury,and NLRP3 knockdown by NLRP3 siRNA can significantly reduce I / Rinjury and improve neurological deficit?2?Nrf2 can inhibit the activation of NLRP3 inflammatory complex by regulation of Trx1 / TXNIP inflammatory complex,thus reducing I / Rinjury.