Mechanism Of SIRT1 In Alleviating Calcific Aortic Valve Disease By Regulating AIM2 Inflammasome

Part1.The role of SIRT1 in Calcific Aortic Valve DiseaseObjective:To investigate the role of SIRT1 in calcific aortic valve disease.Methods:The human study was approved by the ethics committee of the Union Hospital,Tongji Medical College,Huazhong University of Science and Technology.The expression of SIRT1 and RUNX2 in aortic valves were detected by Western blot.The 8-week-old ApoE-/mice and 52-week-old ApoE-/-mice were treated with western diet for 16 weeks.The calcification of aortic valves was detected by von kossa staining,the hemodynamics was detected by doppler ultrasound,and the expression of SIRT1 in aortic valves was detected by immunofluorescence.Human aortic valve interstitial cells(AVICs)were isolated and cultured.The expressions of SIRT1,senescence-related protein P21,osteogenic protein ALP and Runx2 in AVICs of P2 and P9 generation were detected by Western blot,and the formation of calcium nodules was detected by alizarin red staining.Sirt1fl/flCreTaglnApoE-/mice and SirtltgApoE-/-mice were constructed.8-week-old ApoE-/-mice and Sirt1fl/flCreTagln ApoE-/-mice were given western diet for 16 weeks.The calcification of aortic valves was detected by von kossa staining,and the hemodynamics was detected by doppler ultrasound.52 week-old ApoE-/-mice and Sirtltg ApoE-/-mice were given western diet for 16 weeks.The calcification of aortic valves was detected by von kossa staining,and the hemodynamics was detected by doppler ultrasound.Results:In CAVD aortic valves,SIRT1 expression was significantly decreased and Runx2 expression was significantly increased.Aged ApoE-/-mice showed increased calcium nodules in the aortic valves,higher trans valvular peak velocity,and SIRT1 expression was decreased in the aortic valves.The expression of SIRT1 in P9 generation AVICs was significantly decreased,while the expression of P21,ALP and Runx2 was significantly increased.Increasing calcium nodules are formed in P9 generation AVICs.Sirt1fl/flCreTaglnApoE-/-mice showed increased calcium nodules and trans valvular peak velocity,while SirtltgApoE-/-mice showed decreased calcium nodules and trans valvular peak velocity.Conclusion:The expression of SIRT1 in CAVD aortic valves is reduced.Aging can inhibite the expression of SIRT1,while induce the calcification in ApoE-/-mice aortic valves and AVICs.SIRT1 plays a protective role in calcific aortic valve disease.Part2.Mechanism of SIRT1 in CAVD by regulating AIM2 inflammasomeObjective:To explore the mechanism of SIRT1 in alleviating calcific aortic valve disease by regulating AIM2 inflammasome.Methods:After silencing SIRT1 by small interfering RNA in AVICS,transcriptome mRNA sequencing was performed.Data analysis was performed using Dr.Tom analysis platform of BGI.Differential genes were screened and enriched in KEGG database and GO database.The expression of AIM2 in aortic valves was detected by immunofluorescence and Western blot.The expression of AIM2 in the aortic valves of 8-week-old ApoE-/-mice and Sirt1fl/fl CreTagln ApoE-/-mice was detected by immunofluorescence after 16 weeks of Western diet intervention.52-week-old ApoE-/-mice and SirtltgApoE-/-mice were treated with Western diet for 16 weeks,and the expression of AIM2 in the aortic valve of mice was detected by immunofluorescence.SIRT1 in AVICS was silenced by small interfering RNA,and ox-LDL intervention was performed for 72 h.The expression of ALP,RUNX2,and AIM2 was detected by Western blot.After SIRT1 and AIM2 in AVICs were simultaneously silenced by small interfering RNA,ox-LDL intervention was conducted for 72 hours,and the expression of ALP and Runx2 was detected by Western blot.Mitochondrial ROS in AVICs after SIRT1 silencing were detected by flow cytometry and immunofluorescence.The expression of ALP,Runx2 and AIM2 was detected by Western blot after mtDNA intervention AVICs for24 hours.Mito-Tempo,a mitochondrial ROS scavenger,interfered with the AVICs of silencing SIRT1,and Western blot was used to detect the expression of ALP,Runx2 and AIM2.Results:Transcriptome sequencing showed that the silencing SIRT1 in AVICs group had a total of 1797 differential genes(DEGs)compared with the control group.KEGG signaling pathway enrichment analysis showed that the differential genes were highly enriched in NOD-like receptor,Toll-like receptor and NF-?B signaling pathways.Further analysis of NOD-like receptor signaling pathway showed that AIM2 inflammasome gene expression was most significantly upregulated in SIRT1 silencing group.Immunofluorescence and Western blot results showed that AIM2 expression was significantly increased in CAVD aortic valves.Immunofluorescence showed that the expression of AIM2 on the aortic valve of Sirt1fl/flCreTaglnApoE-/-mice was significantly increased,and the expression of AIM2 on the aortic valve of SirtltgApoE-/-mice was significantly decreased.Western blots showed that after SIRT1 silencing,the expressions of ALP,Runx2,and AIM2 in ox-LDL-interfered AVICs were significantly increased.After AIM2 silencing,the expression of ALP,Runx2 was significantly decreased.Flow cytometry and immunofluorescence showed that mitochondrial ROS in AVICS increased significantly after SIRT1 silencing.The expression of ALP,Runx2 and AIM2 was significantly increased after mtDNA intervention with AVIC s.The expression of ALP,Runx2,AIM2 in SIRT1-silenced AVICs was significantly reduced by MITO-TEMPO-mediated intervention.Conclusion:The activation of AIM2 inflammasome is involved in the progression of CAVD.SIRT1 alleviates calcific aortic valve disease by down-regulating mitochondrial ROS mediated AIM2 inflammasome activation.Part3.The role of SIRT1 activator Resveratrol in CAVDObjective:To investigate the role of resveratrol,a SIRT1 activator,in calcific aortic valve disease.Methods:52-week-old ApoE-/-mice were given western diet intervention alone or western diet combined with resveratrol intervention for 16 weeks.Calcium nodules in the aortic valve of mice were detected by von kossa,hemodynamics in the aortic valve of mice were detected by doppler ultrasound,and the expressions of Runx2,SIRT1 and AIM2 were detected by immunofluorescence.The expression of RUNX2,SIRT1 and AIM2 was detected by western blot after RSV pretreatment of AVICs under ox-LDL intervention.Results:Compared with ApoE-/-mice given western diet alone,ApoE-/-mice given western diet combined with resveratrol showed a significant decrease in calcium nodules in aortic valve and a decrease in transvalvular velocity.Immunofluorescence results showed that the expressions of Runx2 and AIM2 on the aortic valve of ApoE-/-mice in the resveratrol intervention group were significantly decreased,while the expression of SIRT1 was significantly increased.Western blot results showed that RSV could up-regulate the expression of SIRT1 in AVICs,while inhibit the expression of Runx2 and AIM2.Conclusion:Resveratrol can activate SIRT1,inhibit the expression of Runx2 and AIM2,reduce the calcification of aortic valve in aged ApoE-/-mice and inhibit the differentiation of AVICs into osteoblastic cells,which has a promising prospect for the prevention and treatment of calcific aortic valve disease.