Mechanism Research Of NF-?B Gene Targeting Transduction In Reducing The Aging Myocardial Ischemic-heart Failure Injury

Objective:This study was designed to investigate the effect of NF-?B signaling pathway during acute myocardial infarction and chronic ischemic heart failure in aged mice.We used cytomegalovirus?CMV?promoter and recombinant single strand AAV9vector and double strand AAV9 vector to validate tissue-specific expression,and screened to obtain better AAV9 vectors which can be used in gene therapy of heart disease.We used IKB??inhibitor of kappa B?,IKB??gene recombinant AAV9 vector for the targeting transduction in aging mouse heart,to investigate whether exogenously introduced IKB?can be used in gene therapy,it can lead to targeted depression of NF-?B,reduction of myocardial infarction,inhibition of myocardial apoptosis,and improve survive of ischemia heart failure in aging mice.The study included:?1?Application of mouse acute myocardial infarction model to investigate the mechanism of NF-?B signaling pathway played in aging mice,to clarify the molecular mechanisms of ageing in acute myocardial infarction and chronic heart failure phase.?2?Via in vivo transduction experiments,to compare baculovirus vector system packaged recombinant vector ssAAV9 and dsAAV9 of CMV promoter,to study the GFP-mediated gene expression and specific time points of expression in mice tissues,to screen the recombinant AAV9 vector which were more suitable for gene therapy of heart disease.?3?Targeted NF-?B gene transduction of aged mice myocardium,via in vivo myocardial acute myocardial infarction model and chronic heart failure model,to investigate the mechanism of IKB?gene overexpression to reduce aging myocardial ischemia injury.Methods:Part ?:To select male C57BL/6J mice with age of 15 to 18-month,toestablish acute myocardial infarction model via left coronary artery liagation.The myocardial infarct size,myocardial apoptosis,IKB?,and the expression of p65 of p50 in cytoplasm and nuclear were observed on day 3 and day 28.Part ?:ssAAV9-CMV-eGFP and dsAAV9-CMV-eGFP vector with the dose of 1×10¹¹vg were injected into C57BL/6J mice via the tail vein,respectively.At 0 week,1 week,2weeks,3 weeks,4 weeks,5 weeks and 8 weeks of viral vector transduction,the laser confocal and Western Blot were used to detect GFP protein expression in heart,liver,spleen,lung,kidney,brain and skeletal muscle.We evaluated the tissue targeting effects of ssAAV9 and dsAAV9 vectors with CMV promoter in vivo.Part ?:15 to 18 month old ageing male C57BL/6J mice were selected,saline,dsAAV9-CMV-eGFP and dsAAV9-CMV-IKB?were injected via the tail,respectively.5weeks later,Western Blot assay was performed to detect myocardial IKB?,p65 and p50expression.5 weeks after in viral transduction,acute myocardial infarction mice model were used,in vivo treatment was the same with the Part I.The myocardial infarct size,apoptosis,the expression of cardiac enzymes CK,CK-MB,LDH,Bcl-2 and Bax were detected.Results:Part ?:?1?It is observed that p65 and p50 positive cells are much higher in MI mice than in SH mice by immunohistochemistry methods?p65 400 vs 154,p50 179 vs 83,P<0.05?.?2?Expression of p65 in MI 3d,7d group in old mice is higher than in SH mice,which means higher activities of NF-?B in mice after myocardial infarction.?3?There were no statistics differences in expression of MMP-2 between SH and MI.Expression of MMP-9is higher in MI than in SH in at 14d?P=0.034 respectively?.?4?Infarct size in MI group was 42.5±6.3%,andapoptosis rate compare with SH were 14.5±2.3%vs 0.7±0.1%,P>0.05.Part ?:?1?Confocal laser scanning and Western Blot were used to detect transduction effeciency of recombinant ssAAV9 and dsAAV9 vectors with CMV promoter at 5weeks,GFP protein was mainly expressed in cardiac and liver,there was almost no expression in spleen,lung,kidney,brain and skeletal muscle.?2?At each detection time point,CMV promoter induced GFP protein expression in the myocardium was significantly higher than that in the liver.GFP protein express was begun in 1 week since AAV9 vectors transduction in myocardium and liver cells.As time extension,the expression gradually increased and transduction reached the peak at week 5.The heart and liver transfection efficiency of dsAAV9 vector and ssAAV9 vector were?90.0±3.2%vs 83.6±3.5%,P<0.01?in heart,and?23.6±3.4%vs 22.2±0.3%,P<0.01?in liver at 5weeks.While the transfection efficiency of dsAAV9 vector and ssAAV9 vector were?75.0±4.2%vs 69.6±3.8%,P<0.01?in heart,and?3.6±0.4%vs 2.4±0.3%,P<0.01?in liver at 8 weeks.It indicated that dsAAV9-CMV-eGFP vector showed stronger target affinity for the myocardial comparing to the ssAAV9-CMV-eGFP vector.It can be steady and efficiently expressed in the myocardium for long-term.While CMV promoter activity can be easily silenced in the liver.Part ?:?1?IKB?gene transduction can efficiently depress aging cardiac NF-?B signaling pathway.When compared between the control group and the empty virus group,the expression of IKB?,p65 and p50 significantly increased?P<0.01?.?2?For in vivo heart research,IKB?gene overexpression can lead to aging myocardial protective effects.The myocardial infarct size in empty virus group,IKB?gene transduction 40.0±7.7%vs34.0±8.0%,the myocyte apoptosis rate was 15.3±2.1%vs 5.1±1.3%.?3?The infarct size and apoptosis rate was statistically significant different when compared with the empty virus group?P<0.01?.It indicated that NF-?B signaling pathway depression can effectively protect the ageing ischemic myocardium from ischemia.The apoptosis related protein Bcl-2/Bax significantly increased,when compared with empty virus group?P<0.01?.?4?In IKB?group,cardiac enzymes of CK,CK-MB,LDH was also significantly reduced,the difference was statistically significant.Conclusion:?1?NF-?B signaling pathway is involved in myocardial infarction and consequence chronic heart failure in aged mice.?2?CMV promoter recombinant dsAAV9 vectors can be expressed in cardiac more efficiently and stably for a long-term comparing to ssAAV9vector.However,the CMV promoter activity can easily be silent in the liver,AAV9-CMV vector is an ideal vector for gene therapy of heart disease.?3?Targeted depression of cardiac NF-?B signaling pathway can stimulate endogenous myocardial damage resistance,so it is a key target for prevention and treatment of the aging myocardial infarction and subsequent heart failure.The IKB?gene can be used as small molecule drugs of gene therapy,it can targeted depress NF-?B signaling pathway,reduce myocardial infarction,inhibit myocardial apoptosis,prevent myocardial ischemia heart failure in aging mice.