Study On The Effect Of Mitofusin2on A7r5Cell Apoptosis

Objective To investigate the effect of mitofusin-2(Mfn2) on rat thoracic aorticsmooth muscle cells (A7r5) apoptosis and the relevant molecular mechanisms.Methods The recombinant plasmid pEGFP-mfn2was transfected into A7r5cellsvia Lipofectamine2000in vitro. A7r5cells were divided into three groups, including control,pEGFP-N1and pEGFP-mfn2. Transfection efficiency was detected by FMC. The expressionof Mfn2in A7r5cells were observed by Western blot when transfected for48h. The effect ofMfn2on A7r5apoptosis was measured by Hoechst Staining, Annexin V-PE/7-AADApoptosis Detection and One Step TUNEL. The expressions of Bcl-xl， Bax,Caspase-9,p-Akt, p-Bad were detected by Western blot. Data was analyzed by ANOVA.Results The transfection efficiencies of pEGFP-N1and pEGFP-mfn2groupreached a peak when transfected for48hours, they were（63.3±2.4）%and（65.2±3.5）%respectively. The difference between the two groups has no significance (p>0.05).The Mfn2protein expression in pEGFP-mfn2group (1.084±0.057) was higher than that of control group(0.534±0.075) and pEGFP-N1group(0.552±0.034)(either P<0.05).Hoechst Staining showedthat the apoptotic cells in pEGFP-mfn2group（13.37±2.85）%were increased compared withcontro（l0.92±0.05）%and pEGFP-N1group（1.33±0.06）%（either P<0.05）. Annexin V-PE/7-AAD Apoptosis Detection showed that the apoptosis rates of pEGFP-mfn2group whentransfected for48hours and72hours were（15.74±2.42）%and（32.23±3.91）%respectively.Almost there was no apoptotic cell in control and pEGFP-N1groups（either P<0.05）. OneStep TUNEL showed that the percentage of late apoptotic cell in pEGFP-mfn2group labeledred fluorescence was （21.3±2.8）%,no apoptotic cell was found in control and pEGFP-N1 group（seither P<0.05）.The Bax protein expression level in pEGFP-mfn2group (0.879±0.126)was higher than that of control group（0.545±0.069） and pEGFP-N1group（0.498±0.074）（either P<0.05）；The Bcl-xl protein expression was decreased in pEGFP-mfn2group(0.527±0.061) compared with control group （0.992±0.107） and pEGFP-N1group（1.011±0.093）（either P<0.05）；The Caspase-9protein expression level was (0.398±0.047)in pEGFP-mfn2group, but it was undetectable in control group and pEGFP-N1group. Thep-Akt protein in pEGFP-mfn2group was (0.572±0.085), which was lower than that ofcontrol group（0.943±0.109）and pEGFP-N1group（0.935±0.072）（either P<0.05）；The p-Badprotein was decreased in pEGFP-mfn2group (0.411±0.046) compared with control group（0.787±0.125） and pEGFP-N1group（0.831±0.114）（either P<0.05）.Conclusions1.Mfn2over expression could accelerate A7r5cell apoptosis.2.Suppressing Ras-PI3K-Akt signaling pathway and activating mitochondrialapoptotic pathway might be the mechanism of Mfn2promoting A7r5apoptosis.