| Background:The NOD-like receptor family,pyrin domain-containing 3(NLRP3)inflammasome is a major intracellular multiprotein complex of the innate immune system and consists of the NLRP3 scaffold,the ASC(PYCARD)adaptor,and caspase-1.Upon stimulation by endogenous and exogenous factors,NLRP3 inflammasomes complete assembly and are activated.This leads to caspase-1 activation and maturation of proIL-1β and proIL-18 into bioactive cytokines,namely IL-1β and IL-18.Both IL-1β and IL-18 then play key roles in initiation and amplification of the inflammatory process.Recent studies have shown that the NLRP3 inflammasome has emerged as a key player in development of cerebral I/R injury.RSV has been reported to protect against cerebral ischemia/reperfusion(I/R)injury in rats,but the underlying mechanism is unclear.Objective:In the current study,we examined whether RSV ameliorates cerebral I/R injury by inhibiting NLRP3 inflammasome-derived inflammation and whether autophagy is involved in this process.In addition,we explored the role of Sirt1 in RSV-mediated protective effects.Methods:⑴ To establish Middle Cerebral Artery Occlusion(MCAO 1hour/R 24hours)in SD rats using Longa suture.RSV was injected at a dose of 100 mg/kg(i.p.)at onset of reperfusion.⑵ 3-MA(600 nmol,10 μg/μL)was injected into the left lateral ventricles 30 min before the MCAO model.After the establishment of MCAO model,RSV was injected at a dose of 100 mg/kg(i.p.)at onset of reperfusion.⑶ Sirt1 siRNA(sense primer 5-GCAGAUUAGUAAGCGUCUUTT-3 and antisense primer 5-AAGACGCUUACUAAUCUGCTT-3)was injected into the left lateral ventricle(15 μL,2 μg/μL).Twenty-four hours after injection of si RNA,the MCAO model was established and RSV was injected at a dose of 100 mg/kg(i.p.)at onset of reperfusion.⑷ In sham、MCAO、MCAO+DMSO and MCAO+RSV groups,the protein expressions of Sirt-1、LC3B、p62、NRLP3 inflammasome、caspase-1、IL1β and IL18 were examined using Western blot technology;the infarct volumes of these groups were assessed by 2% TTC staining;the brain water content and neurological score of these groups were also measured.⑸ In sham 、 MCAO 、 MCAO+DMSO 、 MCAO+RSV and MCAO+RSV+3-MA groups,the protein expressions of Sirt-1、LC3B、p62 and NRLP3 inflammasome were measured using Western blot.⑹ In sham、MCAO、MCAO+DMSO、MCAO+RSV and MCAO+ RSV+Sirt-1 siRNA groups,the protein levels of Sirt-1、LC3B、p62 and NRLP3 inflammasome were examined using Western blot.Results:⑴ After I/R stimulation,levels of Sirt-1,NLRP3 inflammasome,caspase-1,IL-1β,and IL-18 were significantly elevated and autophagy activity was obviously enhanced.However,treatment with RSV clearly inhibited the elevation of NLRP3 inflammasome,caspase-1,IL-1β and IL-18 but further upregulate Sirt-1 expression and enhanced autophagy activity.Meanwhile,RSV treatment could remarkably decreased infarct volume,improve neurological scores and reduce brain water content after 24 h reperfusion.⑵ Pretreatment with 3-MA decreased the ratio of LC3B-II to LC3B-I and increased expression of p62 compared with RSV alone treatment,This indicates that 3-MA treatment can prevent the effects of RSV on autophagy induction.However,3-MA treatment could sobviously blocked RSVmediated inhibition of NLRP3 inflammasome.It is noteworthy that no significant change in protein levels of Sirt1 was observed between the RSV + 3-MA and RSV groups.⑶ The siRNA method was used to reduce expression of Sirt1.Compared with the RSV + MCAO and MCAO groups,pretreatment with Sirt1 siRNA could efficiently decrease protein levels of Sirt1.Subsequently,Sirt1 siRNA effectively diminished autophagy activity relative to RSV or I/R treatment as evidenced by a clear decrease in the ratio of LC3B-II to LC3B-I and an increase in expression of p62.Moreover,RSV-induced downregulation of NLRP3 inflammasomes was remarkably inhibited by Sirt1 knockdown.Conclusion:RSV protects against cerebral I/R injury by inhibiting NLRP3 inflammasome activation through Sirt1-dependent autophagy activity. |
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