Effects Of Paeonol On Bcl-2and Caspase-3Protein Expression After Retinal Ischemia-reperfusion Injury

Objective Establish an animal model similar with the ischemical reperfusion injury ofhuman retina. Intramuscular injection of paeonol is applied to study the morphologicalchange, amount of died nerve cells, died regulation gene Bcl-2, and expression change ofCaspase-3protein of the lab animal with ischemical reperfusion injury in retina, and todiscuss paeonol’s protection and function mechanism upon ischemical reperfusion injuryin retina and its influence to the expression of Bcl-2and Caspase-3protein, thus laying atheoretical foundation for the clinic treatment of retinal ischemic diseases.Method Select54healthy, pure, male, Japanese rabbits, weighted2.5±0.5kg, to divideinto the normal group(6), injury group(24), and treatment group(24) as per the parallelcomparison principle, with the latter two groups divided into6h,12h,24h,48h,72h and120h(5d) subgroups as per different reperfusion time(4rabbits for each time point). For thenormal group, no retinal ischemical reperfusion injury model is made. For the injury andtreatment groups, anterior chamber pressure of the rabbit is boosted by pressurization unitto form retinal ischemia and quickly lower the pressure down to normal level after1h ofischemia, thus presenting the animal model of retinal ischemical reperfusion injury(RIRI).For the treatment group, paeonol is injected via intramuscular way in an amount of5mg/kg,while the normal group and injury group are injected by balanced salt solution. For all thethree groups, the animals will be killed6h,12h,24h,48h,72h and120h after the injection,with their eyes extracted immediately. HE staining method is adopted to observe themorphological change of the retina in different time periods. TUNEL method is used todetect the change of the dying of the nerve cells. Immunohistochemistry is used to detectthe expression change of dying gene Bcl-2and Caspase-3protein. The findings areanalyzed graphically with the data analyzed via SPSS17.0software.Result1morphological change of retina: for the normal group, layers of the rabbit retinaare closely arranged with clear division. For the injury group, slight swell is found in theretina reperfused6h later, with unobvious ganglion cell layer and occasional vacuolardegeneration;12h after the ischemical reperfusion, the swell is worse than6h before, withloose arrangement of the ganglion cell layer;24h after the ischemical reperfusion, theretinal swell is worse, retinal tissues of all layers are loosely arranged, with large loss ofganglion cells and obvious vacuolar degeneration. Inner and outer nuclear layer cells aredisorderly arranged, with obviously thickened inner and outer plexiform layers;48h-72hafter the ischemical reperfusion, the retinal swell is gradually released, with the shape gradually restoring to normal state, but the retinal tissue is generally thinned; and120hafter the ischemical reperfusion, the retinal swell disappears, with the shape normally backto normal state, but the inner layer is shrank and thinned, with cell quantity greatly reduced.The physiological change trend is basically the same with the injury group, but its injuryseverity is easier than the injury group at all time points, with the whole retina thinner thanthe normal state.2Change in the dying of nerve cells: no obvious expression of dying ofcells; for the injury group, the change trend of dying cells can be detected, found after6h,increased after12h, reaching the peak after24h, declining after48h, greatly reducing after72h and no obvious expression found after120h. The change trend of dying cells of theinjury group is basically the same with the treatment group, in which the counting resultsof6h,12h,24h,48h and72h are greatly different, with statistic significance(P＜0.01),while that of120h is of no statistic significance(P＞0.05). Dying cell counting results ofboth the injury group and treatment group at different time points are greatly different(P＜0.01).3Expression of dying gene Bcl-2and Caspase-3protein: nearly no expression ofBcl-2and Caspase-3is found in normal retinal tissue. For the injury group,6h after theischemical reperfusion, expression is found,12h later, the expression increases,24h latter,it reaches the peak,48h latter, it starts to decline,72h latter, it greatly weakens, and120hlatter, no obvious expression is found. Changing trend of the observation indexes of theinjury group is similar with that of the injury group. For comparison between the treatmentgroup and injury group of the same time point, Bcl-2expression obviously climbs(exceptthe120h subgroup), and Caspase-3apparently declines(except the120h subgroup).Counting results of expressed cells of the injury group and the treatment group at differenttime point are apparently different(P＜0.01).Conclusion Paeonol can ease the retinal ischemical reperfusion injury, with a great valuein maintaining the form and function of the retina. Paeonol can retain the dying of retinalnerve cells, possibly related to the improvement of Bcl-2expression and the decline ofCaspase-3expression. It has apparent function in protecting the retinal nerve cells in theretinal ischemical reperfusion injuries.