| Background:Liver generates Angiotensin,transformed into angiotensin ?(Ang ?)in the body.It is widely distributed in human body,especially in the vascular endothelium.Ang ? can produce reactive oxygen species(ROS),accumulation of ROS causing oxidative stress which can cause cell injuring,especially mitochondria in early,and is as the first stage of atherosclerosis in endothelial cell.Oxidative stress can induce endothelial mitochondrial damage and dysfunction.Ang ? as one of the strongest shrinking vascular active substances,is widespread in the vascular smooth muscle.When binding with receptors in the endovascular,Ang ? can be mediated ROS signaling pathways: Rac and P40 moving into the cell membrane,activating the NADPH oxidation reaction and then promoting the generation of ROS.The amount of ROS can increase survival way and enhance the gene expression of resistance of oxidative stress.With the increasing of its concentration,it can induce DNA damage and cell apoptosis.The early injuring of mitochondria is mainly decreasing the regulating and biosynthesis of mitochondrial protein.Mitochondrial transcription factor A(TFAM)which is mitochondrial transcription and replication of the key activation and regulation factor which plays an important role in the oxidative stress.TFAM is produced from cell nucleus,then transferring into the cell mitochondria.As a regulation factor,it can adjust mitochondrial DNA transcription,translation and synthetic after modification.In heart failure,myocardial ischemia-reperfusion injuring by oxidative stress,TFAM as a protective factor against ROS causing mitochondria damage.TFAM is a nuclear gene encoding small molecular peptides,which can maintain the integrity of the mitochondrial DNA apoptosis.When the body attacted by oxidative stress,the expression of TFAM increases quantity,which can lead to increased levels of mitochondria,mitochondrial membrane potential and decreasing levels of apoptosis.Reports that cells attract by oxidative stress,cell of protein kinase A-cAMP-response element binding protein(PKA-CREB)pathways is activated,increasing peroxisome proliferators-activated receptors-1?(PGC-1?),nuclear respiratory factor-1(NRF-1)expression,which further increase the expression of TFAM;TFAM increasing can affinite mitochondrial DNA,regulate mitochondrial DNA copy number,and maintain the integrity and stability of the mitochondrial DNA,promoting the mitochondrial DNA damage repair.However,Oxidative stress abnormal accumulation can be damaged the function of the nucleus DNA,resulting in the expression of TFAM decreased.A study shows that it may be associated with the negative feedback.ROS stimulates cell,leading to increasing the level of mitochondrial TFAM to intranuclear TFAM combining with NRF-1 to form immune coprecipitation,which reducing the expression of NRF-1 quantity and then decreasing the cytoplasm of TFAM expression.At the same time,studying shows that in the cytoplasm of Lon proteolytic enzymes make phosphorylate TFAM crack and high concentration of ROS inhibit the HSP60-70-TFAM-Lon complex formation to inhibiting the biosynthesis of mitochondria.Therefore,this study aims to explore the different concentrations of Ang-? on the umbilical vein endothelial cells,to observate TFAM expression in endothelial cells and cell function,then to further for the early detection and prevention of atherosclerosis.Objective: To observe the changes of mitochondrial transcription factor A(TFAM)expression and vascular endothelial cells influenced by Angiotensin ?(Ang-?).Methods:1.Cells:Human umbilical vein endothelial cells(HUVECs)were from ScienCell Compony.2.Experimental group and the intervention:Collection of logarithmic phase HUVEs cells,cultivated 12 hours with 1 x106/mL.Cells were treated by 0?10-7?10-6?10-5mol/L Ang-?(group A?B?C?D).The experimental group were cultivating without serum culture medium.3.Westeren-blot:the expression of TFAM in each group were detected by Westeren-blot.4.RT-PCR:the expression of TFAM in each group were tested by RT-PCR.5.Flow cytometry instrument: each groups were monitored mitochondrial membrane potential and the cell apoptosis rate by flow cytometry instrument.6.The statistical analysis was performed with SPSS19.0,Data were presented as mean±SD.Differences were analyzed by analysis of variance.The statistical significance of differences was accepted at the level of P<0.05.Result:1.Different concentrations of Ang-? on HUVECs TFAM protein,TFAMmRNA expressionThe effect of Ang-? on TFAM expression was detected at the gene level and protein level:under the action of different concentrations of Ang-?(0,10-7,10-6,10-5mol/L),each group of cell by Ang-? cultured for 24 h.Westeren-blot test showed that the expression in each group were 0.94±0.04?0.78±0.09?0.48±0.09?0.40±0.07.With the concentration of Ang-? increasing,the TFAM protein expression rising,which showed a dose-dependent tendency.The difference was significant by analysis of variance(P<0.01).The culture supernatant level of TFAM mRNA was detected by RT-PCR were 0.94±0.08?0.78±0.09?0.44±0.07?0.40±0.08,the assays showed that with the concentration of Ang-? increasing,the level of TFAM mRNA was getting higher and higher,rendering a dose-dependent relationship.The analysis of variance indicated the difference was significant(P<0.01).2.Each cell membrane potential detection JC-10 forms polymer in the mitochondria and emits red light with fluorescence;Before the early apoptosis cells,mitochondrial membrane potential performances decline and namely JC-10 exists monomer emitting green fluorescence in the cytoplasm.Different concentrations of Ang ? cells cultivating 12 hours,A,B,C,D group of mitochondrial membrane potential to reduce cell levels(%)was 26.98±4.46?41.74±4.24?60.47±4.52?70.16±3.07.Comparing experimental group with control group increased mitochondrial membrane potential rate of decline increased,and the difference was statistically significant(P < 0.01).3.Cell apoptosis detectionDifferent concentrations of Ang-? cultivating,each group of early apoptosis rate and late apoptosis rate were 4.0%,4.0%,10.8%,2.9%,5.2%,8.2%,1.4%,15%.In group B,early apoptosis more increased than group A.With the increase of concentration of Ang – ?,early apoptosis reduce,late apoptosis increase.Conclusion:1 Ang ? decreased HUVECs expression of TFAM2 Ang ? increased the rate of declining mitochondrial membrane potential3 Ang ? increased the rate of cell apoptosis... |
PDF Full Text Request