The Protective Effect Of Praeruptorin A On Cerebral Ischemia Reperfusion In Rats By Regulating The Expression Of Bcl-2/Bax, Caspase9 And Caspase3

[Objective]Based on the previous research, praeruptorin A on cerebral ischemia / reperfusion in rats has a protective effect.Brain neuronal injury following cerebral ischemia reperfusion showed cell necrosis and apoptosis.Apoptosis is regulated by many kinds of gene expression, such as Caspase family and Bcl-2 family, which are the important regulatory genes of apoptosis.Through the experiments of praeruptorin A on cerebral ischemia reperfusion injury in rats after the detection effect on Bcl-2/Bax, caspase9, Caspase3;we can study of praeruptorin A whether through inhibiting cell apoptosis and protect the brain of cerebral ischemia and reperfusion injury. [Material and methods]Buy 96 SPF grade healthy SD rats, weight 240-260 g, take the random method is divided into 8 groups, 12 only a group,divided into normal group, sham operation group, normal saline group, solvent control group, nimodipine group(1mg/kg), 1mg/kg Pd-Ia group, 5mg/kg Pd-Ia group, 10mg/kg Pd-Ia group.The laboratory rat Middle Cerebral Artery Occlusion made by suture method in ischemia rat model.In each group, 4 rats were randomly selected, and MCAO method was used to create the animal model of cerebral stroke.After the modeling, at 3h after ischemia reperfusion, 6h after reperfusion, 12 h after reperfusion, 24 h after reperfusion, the reperfusion after administration of 48 h, 72 h after reperfusion(a total of 6 times for Drug Administration).And then the rats were sacrificed and the brains and slices were taken and TTC staining was carried out.Randomly selected 4 rats in each group of the 8 groups, using the MCAO method to make the model, after the success of the model,open perfusion at the same time after ischemia 3h,after reperfusion, 12 h, 6h, and 24 h after reperfusion,a total of 4 drugs.The next, the brain, fixation, dehydration, paraffin embedded,then to detect the apoptosis was detected by immunohistochemistry and Tunel method.4 randomly selected in each group of 8 again,after the MCAO method was used to build the model successfully.Open perfusion at the same time after ischemia 3h,After reperfusion, 12 h, 6h, and 24 h after reperfusion,a total of 4 drugs,Then the mice were killed and the rats were broken,the cerebral cortex was selected for WB experiment and RT-PCR experiment.All the results were expressed by mean plus or minus standard deviation(x±s).Statistical analysis of the data obtained with SPSS 22 statistical software,single factor variance analysis was used to analyze the difference between groups, P<0.05 said there are differences.If it is a non normal distribution,we choose non parametric test,P<0.05 said there are differences.[Results]1.Study on the biological behavior, we can find that after the MCAO model was made, the biological behavior score of the rats was 1, 2 and 3, which indicated that the model was successful.In the sham operation group and the normal group, the biological behavior score was 0.The rat model of MCAO was made,after awakening, the scores and 24 h were compared,the score after 24 h was lower in the Pd-Ia experimental group than in the wake,P<0.01, there are significant differences.Score after 24 h,comparison of Pd-Ia experimental group and physiological saline group,the scores of Pd-Ia in the experimental group were lower than that in the normal saline group.Comparison of Pd-Ia1mg/kg group and physiological saline group,P<0.01, there are significant differences.2.After Tunel detection,cerebral ischemia reperfusion injury can be found in the presence of apoptotic cells,through the use of praeruptorin A, the number of apoptotic cells decreased.Among them, the 5mg/kg Pd-Ia group was the most significant reduction in apoptosis.3.Immunohistochemical results show,24 hours after cerebral ischemia and reperfusion, Bcl-2 was expressed.After using Pd-Ia, the expression of Bcl-2 was significantly increased.Pd-Ia three different doses of the experimental group and the physiological saline group, the solvent group, nimodipine group, the difference was significant.Pd-Ia of the three different dose groups compared with each other, the difference was significant.Among them, the 5mg/kg Pd-Ia group could significantly promote the expression of Bcl-2.The visible expression of immunohistochemical detection of activated Caspase3,Caspase3 after cerebral ischemia reperfusion were significantly activated.Comparison between the saline group and solvent group, nimodipine group and praeruptorin A three different doses of the experimental group, We can find the praeruptorin A group can significantly reduce the activation of type Caspase3. Among them, the inhibitory effect of 5mg/kg Pd-Ia group was the most obvious.4.From the Western blot a we can find,5mg/kg Pd-Ia group can significantly inhibit the activation of caspase9 Caspase3.5mg/kg Pd-Ia group compared with the normal saline group, p<0.01, there were significant differences.Praeruptorin A experiment group compared with normal saline group, the ratio of Bcl-2/Bax increased, p<0.01, there was significant difference.5.From the PCR experiment,Bcl-2/Bax experimental group compared with the normal saline group, the Pd-Ia ratio was significantly increased, p<0.01, there were significant differences. [Conclusion]1.Praeruptorin A can change animal behavior improvement after MCAO,reduce infarct volume,inhibition of apoptosis of rat brain cells.2.The protection of praeruptorin A on cerebral ischemia reperfusion injury possible through inhibiting apoptosis pathway to achieve3.The effect of cerebral ischemia reperfusion injury in 5mg/kg Pd-Ia group was the best in the three experimental groups of praeruptorin A.