| Background and ObjectInflammation plays an important role in the pathogenesis process of coronary atherosclerosis heart disease.Atherosclerosis is an inflammatory process involved in inflammation mediators,such as variety of adhesion molecules.Inflammation reaction in endothelial cell injury is the major contributor for the initiation and progression of atherosclerosis.Emerging evidences suggest that human nicotinamide adenine dinucleotide dependence to histone acetylation enzyme3(SIRT3) plays an important role in energy metabolism,signal transduction,myocyte hypertrophy and longevity.However,the effect of SIRT3on endothelial cell injury in atherosclerosis process has not been reported at home and abroad.Therefore,this study is to investigate the regulation relationship between human SIRT3gene and inflammatory mediators in the endothelial cell injury process,and to further reveal the SIRT3gene effect in atherosclerosis development process.Methods1. Screening interference sequences(1) Cultivation human aortic endothelial cells:Human aortic endothelial cells were adherent cultured in nutrient solution including endothelial cell medium(ECM) supplemented with1%endothelial growth factor (EGF),5%fetal bovine serum(FBS) under the condition of37℃and5%CO2and the third-seventh generation cells in good state were used for experiments.(2) Grouping and transfection:The cells were randomly divided into blank control group,negative control group,transfection reagent control group and SIRT3siRNA (1,2,3,4) group.When80%cells were fusion together,we went to transfect.The transfection reagent was hiperfect.In transfection reagent control group,transfection only added transfection reagent without interference sequence.At transfection time,transfection reagent control group was just added transfection reagent without interference sequence.Negative control group was added transfection reagent and negative control sequence and transfection reagent.We tested relevant indexs after transfection for24h.(3) Cell morphological change was observed with microscope.(4) Fluorescent protein expression in negative control group was observed with fluorescent inverted microscope.(5) SIRT3mRNA in each group was tested by qRT-PCR.(6) SIRT3protein expression in each group was tested by western blotting.2.Expressions of SIRT3mRNA,ICAM-1and E-selectin protein in HAEC stimulated by ox-LDL after interference SIRT3gene(1) Experiment was randomly divided into blank control group,siRNA group,(20,30,40μg/mL) ox-LDL group and siRNA+(20,30,40μg/mL) ox-LDL group.Ox-LDL groups were stimulated for6h by different concentration ox-LDL.The siRNA was the best selected SIRT3interference sequence.All siRNA+ox-LDL groups were stimulated for6h by ox-LDL after transfection24h.(2) SIRT3mRNA was detected by qRT-PCR.(3) Mean fluorescence intensities (MFI) of ICAM-1and E-selectin antibody were detected by flow cytometry.Results1.Results of screening interference sequences(1) Compared with the blank control group,cells in each SIRT3siRNA group exhibited morphological alterations such as volume increase,homogeneity decrease and intercellular space enlargement with optical microscope.These features were obvious in SIRT3siRNA2group.(2) Under the same view in optical microscope,more than90%cells showed green fluorescence in negative control.(3) Compared with control groups,the SIRT3mRNA expression in each SIRT3siRNA group reduced significantly in different extent(all P<0.05).The interference effect in SIRT3siRNA2group was obvious, and its silent efficiency was88.616%.(4) Compared with all control groups,relative grey value in four SIRT3siRNA groups were different significantly(all P<0.05).The interference effect in SIRT3siRNA2group was obvious,and its silent efficiency was80.372%.2.Expression of SIRT3mRNA,ICAM-1and E-selectin protein by different ox-LDL stimulated after SIRT3gene interference.(1) Compared with the blank control group,SIRT3mRNA relative expression was different in every ox-LDL concentration group (all p<0.05);and with increase of ox-LDL concentration,SIRT3mRNA expression significantly reduced(all P<0.05).SIRT3mRNA relative expression in siRNA group significantly less than that in the blank control group(P<0.001).Compared with siRNA group,SIRT3mRNA relative expression in all siRNA+ox-LDL groups had no statistical difference (all p>0.05).(2) Compared with the blank control group,MFI of ICAM-1in siRNA group and ox-LDL concentration groups had no statistical difference (all p>0.05).Compared with siRNA group,MFI difference of ICAM-1in each siRNA+ox-LDL group was statistically significant (P<0.05);with increase of ox-LDL concentration,MFI of ICAM-1in siRNA+ox-LDL group significantly increased in turn (all p<0.05).(3) Compared with the blank control group,MFI of E-selectin in siRNA group and20,30μg/mL ox-LDL concentration groups had no statistical difference(all P>0.05).However,MFI of E-selectin inceased significantly in40μg/mL ox-LDL group(P<0.05).Compared with siRNA group,MFI difference of E-selectin in each siRNA+ox-LDL group was statistically significant(P<0.05);with increase of ox-LDL concentration,MFI of E-selectin in siRNA+ox-LDL group significantly increased in turn(all p<0.05). Conclusion1.This experiment has screened successfully SIRT3small interference RNA sequences for further study.Accurate silence SIRT3gene can provide convenience for research the relationship between SIRT3and other genes.2.Through this experiment,we find that SIRT3gene expression increases significantly in human aortic endothelial cells stimulated by low concentration ox-LDL,but SIRT3expression decreases with high ox-LDL concentration.These findings show that SIRT3may be as a protection factor has compensatory function,but its compensatory effect is subject to ox-LDL concentration.3.SIRT3gene was associated with expression of E-selectin and ICAM-1in human aortic endothelial cells stimulated by ox-LDL.4.SIRT3may be an essential molecule to promote the repair of damaged endothelium cell and to inhibit the progression of atherosclerosis,but the mechanisms involved in the present findings needs further research. |
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