Farrerol Alleviates Myocardial Ischemia/Reperfusion Injury By Inhibiting NLRP3 Inflammasomein Macrophages

BackgroundSterileinflammatory injury is one of the main pathogenesis of myocardial Ischemia/reperfusion(I/R)injury.Endogenous damage-associated molecular patterns(DAMPs),passively releasing during myocardial damage and adenosine triphosphatase(ATP)from reperfusion,leading to activation of the NLRP3 inflammasome,inducing activation of Caspase-1.The main role of Caspase-1 is to cleave inactive pro-IL-1β and pro-IL-18 into mature and functional IL-1β and IL-18,or trigger cell pyroptosis,amplifying the inflammatory response.Farrerol(FA),naturally found in Rhododendron dauricumL.leaves,is a dihydro flavonoid active substance with antibacterial,antiinflammatory,antioxidant,and other biological activities.FA has been reported to play an organ protective role by reducing IL-1β and IL-18 secretion in a variety of inflammatory disease models.Based on the above information,we supposed that FA might regulate the activation of NLRP3 inflammasome in macrophages and relieve myocardial I/R injury.Methods(1)Fifty C57BL/6J male mice of 8-week-age were divided into 5 groups,including ①Sham group,②I/R,③I/R+FA(10 mg/kg/day),④I/R+FA(40 mg/kg/day),and ⑤FA(40 mg/kg/day).I/R mice models were established via 30 min of ischemia and subsequent 24 h of reperfusion of left anterior descending artery.The Sham group only received simple skin incision.All groups received FA treatments,C57BL/6J mice were intraperitoneally injected with FA for 7 days before I/R and the same volume of normal saline was used as control.Heart function was measured by transthoracic echocardiography.HE,TUNEL and Evans blue/the 2,3,5 triphenyltetrazolium chloride(TTC)double staining to detected the severity degree of myocardial injury and infarct size respectively.CK-MB,LDH,Troponin-Ⅰ,NT-proBNP,IL-1β,IL-6,TNF-α,GSH-Px,SOD,LPO and MDA were detected with commercial kit.Western blot was used to detect the expression of NOX4 and apoptosis-related proteins,such as caspase3,PARP1,Bcl2 and Bax.(2)Mice in a C57BL/6J background deleting Nrf2(Nrf2-/-)or Nlrp3(Nlrp3-/-)were purchased,or wild-type(WT)mice were injected 3methyladenine(3-MA)or clodronate liposomes to explore whether FA might play a protective role in themyocardium through the Nrf2,NLRP3,autophagy or macrophagedependent pathway.(3)Inflammasome activation test and co-immunoprecipitation was conducted in bone marrow-derived macrophages(BMDMs)of mice to explore the molecular mechanism of FA regulating NLRP3 activation.ResultsPart Ⅰ FA can relieve I/R induced myocardial injury in miceFA preconditioning(10 mg/kg or 40 mg/kg)can relieve myocardial injury,reduce infarct size and improved heart function of mice treated with myocardial I/R.FA can significantly decrease the levels of pro-inflammatory cytokines(IL-1β、IL-6 and TNF-α).In addition,FA can alleviate oxidative stress andapoptosis in myocardial I/Rtreated mice.Part Ⅱ FA inhibits NLRP3 inflammasome activation in macrophages by blocking NLRP3-NEK7 binding(1)The I/R-induced myocardial injury was more severe in Nrf2-/-mice than in that of WT mice.However,the absence of Nrf2 does not reverse the protective role of FA againstI/R-induced myocardial injury.(2)After deleting autophagy with 3-MA,FA still alleviated I/R-induced myocardial pathological injury.(3)After macrophages were removed by clodronateliposomes,HE staining indicated that there was no significant difference in myocardial pathological injury and serum markers of myocardial injury(CK-MB,LDH,Troponin-1 and NT-proBNP)between I/R+FA(40 mg/kg)and I/R treated group.Likewise,in Nlrp3-/-mice,the protective role of FA against I/R-Induced myocardial pathological injury was eliminated.(4)Western blotting indicated that FA significantly decreased the protein expression of NLRP3,caspase-1,IL-1β,and proIL-1β in heart tissue of WT mice,but did not alter the level of the pro-caspase-1.(5)In vitro,FA inhibited the secretion of IL-1β and caspase-1 in macrophages,but did not affect the level of pro-IL-1β,pro-caspase-1 and NLRP3.Co-immunoprecipitation experiments indicated that FA inhibited the activation of the NLRP3 inflammasome in macrophages by directly irreversible blocking the NLRP3-NEK7 interaction,rather than disturbing NLRP3-ASC,NLRP3-NLRP3 interaction.ConclusionsFA plays a protective role against myocardial I/R injury in mice.This pharmacological effect is associated with the inhibition of the NLRP3 inflammasome in macrophages by irreversible blocking NLRP3-NEK7 interaction,but not through Nrf2 or autophagy-dependent pathway.