Role Of Targeted Inhibition Of NF-κB Signaling Pathway In Myocardial Ischemia-reperfusion Injury In Aged Mice

Objectives:The purpose of this study was to explore the effect of age on myocardial ischemia-reperfusion injury,and to investigate the mechanisms of NF-κB signaling pathway in the increased susceptibility of aging myocardial ischemia-reperfusion（I/R）injury.Recombinant AAV9 vector carring IκBα（inhibitor of kappa B,IκBα）gene was transfected into senescent cardiomyocytes or aged hearts to identify whether IκBαcould be regarded as a gene therapy drug by suppressing the NF-κB pathway,reducing myocardial infarction and decreasing programmed cell death（PCD）following ischemia-reperfusion injury in vitro or in vivo.The main research contents include:1)Establishment of mouse I/R injury model,to compare the differences of myocardial injury between young and aged mice,and to clarify that excessive activation of NF-κB signaling pathway was accounted for the increased susceptibility of myocardial I/R injury in old mice;2)In vitro,isolation and cultivation of the primary senescent cardiomyocytes induced by D-galactose.Then,transfected the senescent cardiomyocytes with dsAAV9-CMV-e GFP（dsAAV9-e GFP）and dsAAV9-CMV-IκBα^S32A,S36A（dsAAV9-IκBα）respectively.Application of hypoxia/reoxygenation（H/R）model,to identify whether overexpression of IκBαgene can prevent H/R-induced cardiomyocyte injury and explore its possible molecular mechanism;3)In vivo,targeted delivery of IκBαgene to heart in aged mice through the tail vein injection,to observe its effect on the prevention the myocardial I/R injury and to clarify the its molecular mechanism.Methods:Part Ⅰ:Establishment the I/R model in male C57BL/6J mice aged 3 months and 18months.TTC+Evans blue double staining method was used to detect myocardial infarct size.Serum myocardial enzymes（LDH,CK-MB）,inflammatory factors（TNFα,IL-6,IL-1β）and oxidative damage（MDA,SOD）indicators were assessed.Cardiac ultrasound Vevo（?）3100 was used to detect cardiac function after I/R at 7 days,14 days,28 days.Western blot was applied to detect the expression of NF-κB signaling pathway proteins and programmed cell death-related proteins（Bax,Bcl-2,GSDMD,Beclin-1）.Part Ⅱ:In vitro study,we isolated and cultured neonatal rat ventricular cardiomyocytes（NRVMs）,and then induced senescent cardiomyocytes with 10g/L D-galactose.The dsAAV9-e GFP and dsAAV9-IκBαvirus were transfect to senile cardiomyocytes,respectively.After establishing H/R model,flow cytometry was used to detect myocardial cell apoptosis or proptotic proportion,Hochest/PI staining to detect proptotic rate,JC-1 to detect changes in mitochondrial membrane potential（Δψm）,enzyme-linked immunosorbent assay（ELISA）to detect the levels of inflammatory factor such as interleukin-1 beta（IL-1β）,and the MDA and SOD contents were measured to assess the oxidative injury.Immunofluorescence and Western blot were used to detect protein expression of NF-κB signaling pathway,inflammasome（NLRP3）,and pryoptosis（Caspase-1,GSDMD,IL-1β）,autophagy（Beclin-1,LC3Ⅰ/Ⅱ）and apoptosis（Bax,Bcl-2）.Part Ⅲ:18-month-old C57BL/6J male mice were injected with dsAAV9-e GFP and dsAAV9-IκBαvirus in the tail vein for 5 weeks,and then an I/R model was established to measure myocardial infarct size,myocardial apoptosis,leukocyte and macrophages infiltration by CD45 and CD68 labeled antibodies.Serum was taken to detect myocardial enzymes（LDH,CK-MB）,inflammatory factors（TNFα,IL-6,IL-1β）and oxidative damage（MDA,SOD）.Vevo（?）3100 was used to detect the short-term and long-term cardiac function.We detected the protein expression of NF-κB pathway,inflammasome（NLRP3）,pyroptosis（Caspase-1,GSDMD）,apoptosis（Bax,Bcl-2）and autophagy-related proteins（Beclin-1,LC3Ⅰ/Ⅱ）.Results:Part Ⅰ:1)Western blot identified that IκBαcould successfully transfected in aged heart and inhibited nuclear translocation of p65 subunit;2)Compared with the young group,aged mice had a larger myocardial infarct size（57.89±2.55%vs 45.92±2.63%,P<0.05）but lower ejection fraction（21.28±3.11 vs 31.60±3.71,P<0.05）.Serological tests revealed that myocardial enzymes,oxidative damage,and inflammatory factor levels in the aged group further increased than that in the young group Increase（P<0.05）;3)Western blot results showed that cytoplasmic proteins IκBα,p65,and p50 decreased,while nuclear proteins p65 and p50 increased significantly in a time-dependent manner,suggesting that nuclear translocation of NF-κB signals occurred was activated in both ischemia and reperfusion process.Compare with the young group,the activation of NF-κB was more robust in aged mice（P<0.05）;4)Compared with the young group,the Bax/Bcl-2 ratio and expressions of Beclin-1 and GSDMD-N were significantly elevated in aged group（P<0.05）,indicating severe PCD occurred in aged hearts.Part Ⅱ :1)After transfected with dsAAV9 virus to senile cardiomyocytes for 5 days,the intensity of GFP reached 70%,and the expression of GFP and IκBαprotein increased significantly（P<0.05）,indicating AAV9 could be successfully transfected into primary cardiomyocytes;2)Compared with the normal group,the cell viability,SOD,andΔψm of senile cardiomyocytes decreased significantly after H/R treatment,while the supernatant LDH and IL-1β,the cellular MDA content significantly increased,which could be reversed by IκBαtransfection（P<0.01）;3)The apoptosis rate was measured by flow cytometry.The results showed that the ratio of Annexin V-PE+/7-AAD-cells in the normal control group was 4.78±0.78%,and that in the H/R control group and GFP group were 37.33±1.32%and 39.83±0.45%,while it was decreased to 21.370±4.04%in the IκBαgroup（P<0.05）,indicating that IκBαcan resist H/R-induced cardiomyocyte apoptosis.Meanwhile,the ratio of Annexin V-PE+/7-AAD+cells in the normal control group was 9.63±0.39%,in the H/R control group and the GFP group were 18.57±2.99%and 18.13±1.92%,respectively,while the it decreased to 7.56±2.40%in the IκBαgroup（P<0.05）,suggesting that IκBαtransfection can resist H/R-induced pyroptosis in senile cardiomyocytes;4)Hoechst/PI staining further evaluated cell pyroptosis.The proportion of PI staining positive cells in the normal group was 1.83±0.38%,the H/R control group and the GFP group were 42.37±3.06%and 41.53±2.09%,respectively,while the ratio in IκBαgroup decreased to 17.90±2.01%（P<0.05）,suggesting that IκBαcan resist H/R-induced myocardial cell pyroptosis;5)Immunofluorescence and Western blot identified that IκBαsuccessfully inhibited nuclear translocation of p65 subunit;6)H/R significantly upregulated the expression of PCD-related proteins,while IκBαtransfection reduced apoptosis by lowering the Bax/Bcl-2 ratio,decreased autophagy by down-regulating the ratio of Beclin-1 and LC3Ⅱ/Ⅰ,and inhibited pyroptosis by suppressing the NLRP3/Caspase-1/GSDMD axis in senile cardiomyocytes exposed to H/R.Part Ⅲ:1)Western blot identified that IκBαcould successfully transfected in aged heart and inhibited nuclear translocation of p65 subunit;2)Compared with GFP group,IκBαgene could reduce the myocardial infarct size（56.92±3.22%vs 26.33±1.54%,P<0.01）and cardiomyocyte apoptosis index（26.67±1.65%vs 11.83±0.95%,P<0.01）in aged mice after I/R treatment,indicating that IκBαoverexpression can effectively protect aged hearts from I/R injury;3)Immunofluorescence results showed that IκBαcould significantly reduce the infiltration of leukocytes and macrophages,decrease myocardial enzymes and inflammatory factors（TNFα,IL-6,IL-1β）,increase SOD activity,and reduce MDA content after I/R injury in aged mice（P<0.05）,suggesting that IκBαhas the effect on preventing inflammation and oxidative damage against I/R injury in aged mice;4)Western blot results show that IκBαoverexpression can down-regulate the expression of autophagy,apoptosis and pyroptosis-related proteins,suggesting that IκBαreduced I/R induced myocardial loss by inhibiting programmed cell death;5)Compared with the GFP group,the IκBαgene significantly reduced the left ventricular end-systolic area（LVESA）,left ventricular end-systolic volume（LVESV）,Left ventricular end-diastolic area（LVEDA）,Ejection fraction（EF）and Fractional area change（FAC）at 7 and 14 days after I/R（P<0.05）,but this protective effect failed at 28 days（P>0.05）,indicating that IκBαcan effectively reduce the ventricular enlargement and improve the cardiac function in aged mice.Conclusions:1)Endogenous myocardial injury is exacerbated companying an increase of autophagy,apoptosis,and pyroptosis after I/R in aged mice,Excessive NF-κB signaling pathway may mediate ischemia-reperfusion injury in old myocardium and be the key target of age heterogeneity;2)In vitro,transfection of IκBαgene reduces oxidative damage and inflammatory response by inhibiting NF-κB signaling pathway,reduces autophagy level,stabilizes mitochondrial function,decreased cardiomyocyte apoptosis and pyroptosis,and ultimately Improve the vitality of cardiomyocytes;3)In vivo,targeted transfection of IκBα,as a small molecule gene therapy drug,can effectively inhibit activation of the NF-κB signaling pathway,ameliorate inflammation and oxidative damage,reduce programmed cell death（apoptosis,pryroptosis,autophagy）,alleviate the myocardial infarct size,and improve cardiac function in aged mice,which may provide a new strategy for aged myocardial ischemia/reperfusion injury.