The Effect Of Silencing PBR Expression On Cardiomyocytes Subjected To Anoxia-reoxygenation Injury

Objective:We established the anoxia-reoxygenation (A/R) model of H9C2cardiomyocytes in order to investigate the effect of PBR which is located in the mitochondrial outer membrane subjected to anoxia-reoxygenation injury. And it may offer new ways and experimental basis to further explore the mechanism of ischemia-reperfusion injury and look for myocardial protection drugs.Methods:The expression of PBR was assayed using RT-PCR and Western Blot analyses after processing A/R, APC;To PSES-HUS plasmid as the carrier, construct PSES-PBR plasmid; We established the low expression levels of PBR protein in H9C2cells by gene transfection technique and RNA interference, The H9C2cells were randomly divided into four groups:(1) Control group;(2) A/R group;(3) PSES+A/R group;(4) PSES-PBR+A/R group. Every group repeats six times.Cell viability was detected by MTT method. SOD and GSH-Px in H9C2cells were measured by a colorimetric method. Lipid peroxide MDA in the culture medium of H9C2cells was analyzed with an automatic biochemical analyzer. The percentage of apoptosis was measured by annexin V-FITC/PI double staining with flow cytometry,intracellular ROS and the mitochondria membrane potential were also measured with flow cytometry. Mitochondrial swelling was assayed by spectrophotometer.Results:1.The expression of PBR mRNA and protein after processing A/R were significantly increased compared to normal H9C2cells but after processing APC were significantly decreased compared to A/R using RT-PCR and Western Blot analyses.2. We successfully constructed an siRNA PBR expression recombinant vector which was identified by DNA sequencing. The expression of PBR protein which transfected with recombinant vector were significantly decreased compared to normal H9c2cells using Western Blot analyses.3. After anoxia/reoxygenation, MDA, ROS, mitochondrial swelling and apoptosis were remarkably increased while activities of SOD, GSH-Px, cell viability and the mitochondria membrane potential were decreased in H9C2cells compared with control group (p<0.01).However, in the PSES-PBR+A/R group, MDA,ROS, mitochondrial swelling and apoptosis were remarkably decreased while activities of SOD, GSH-Px, cell viability and the mitochondria membrane potential were increased in H9C2cells compared with A/R group (p<0.01). PSES+A/R group and A/R group compared each index has no significant difference (p>0.05).Conclusion:Silencing PBR has played an important role in protecting against anoxia/reoxygenation injury by reducing oxidative stress and subsequently maintaining the stability of mitochondrial membrane potential, preventing the open of mitochondria permeability transition pore.