| Objective: To investigate the expression changes of caspase-3,Bcl-2 and Bax in cerebral ischemia reperfusion(MCAO/R)after cerfain-1 in rats.Methods: Seventy SD male rats were randomly divided into sham operation group(n=20 in Sham group),operation group(n=25 in I/R group),and pretreatment group(nesfatin-1+I/R group n =25).Experimental animal models were made using the modified Zea-Longa method.After modeling,the lower limb exercise,ie behavior,was scored according to the Longa score.The degree of neurological damage was determined by MCA/R rats by the length of the longa score and the stability of the successful model was determined.For the sham group,only the vascular separation,I/R group,nesfatin-1+I/R group were sacrificed for 2 hours and then reperfused for 24 hours.The rats were sacrificed by decapitation.The brain tissue was protected from light at 37 °C in water,0.2% TTC.The stain was stained,fixed in 4% paraformaldehyde for 24 hours,and the percentage of cerebral infarction volume in different groups was measured.After the modeling of each group was stabilized,the rats in each group were killed by decapitation and the brain tissues were fixed or cryopreserved.Morphological changes of brain tissue were observed by hematoxylin-eosin staining.The morphology of cortical and hippocampal neurons was observed by Tunel method.The neuronal apoptosis index was counted.The brains of each group were detected by immunohistochemistry and WesternBlot technique.The expression changes of tissues caspase-3,Bcl-2 and Bax were statistically significant.Results: 1.The results of TTC staining showed that the percentage of infarct volume in the brain of sham group,I/R group and nesfatin-1+I/R group increased first and then decreased,I/R group and nesfatin-1+I/R group.The volume of cerebral infarction was larger than that of sham group(p<0.05).Compared with I/R group,nesfatin-1+I/R group: human recombinant protein nesfatin-1 has protective effect on cerebral ischemia reperfusion,and the volume of cerebral infarction is reduced.(p<0.05).2.Hematoxylin-Yinhong staining results showed that the brain tissue fixation,dehydration,embedding and staining showed that there were no abnormalities in the number and size of neurons in the sham group,no swelling of the neurons was observed,and no capillary was seen.Hyperplasia,hemorrhage,no looseness in the cerebral cortex.The results of brain tissue staining in I/R group showed that degeneration and necrosis of nerve cells,formation of softening lesions,slight proliferation of glial cells,widening of neuronal fissures,powdery flocculation in the fissures,and subarachnoid congestion.The nesfatin-1+I/R group was compared with the I/R group: the human recombinant protein nesfatin-1 reduced neuronal degeneration and necrosis;reduced neuronal edema.3.The results of apoptosis detection in Tunel cells showed that the nucleus squeezing and brown-yellow granules in the cortex and hippocampus were increased in the I/R group and the nesfatin-1+I/R group compared with the sham group.The statistical results showed that the neuronal apoptosis index of I/R group and nesfatin-1+I/R group was higher than that of sham group(p<0.05).The nesfatin-1+I/R group was compared with the I/R group: the nucleus of the cortical and hippocampal neurons was reduced,the brown-yellow particles were decreased,and the neuronal apoptosis index was decreased(p<0.05).4.Immunohistochemistry showed that the results of sham group showed that caspase-3 and Bax were negative in brain tissue and Bcl-2 was strongly positive in brain tissue.The results of I/R group showed that caspase-3 and Bax were strongly positive in brain tissue and Bcl-2 was weakly negative in brain tissue.The results of nesfatin-1+I/R group showed that the expression of caspase-3 and Bax in brain tissue was weakly positive;Bcl-2 was positive in brain tissue.The statistical results showed that the expression of caspase-3 was higher in the I/R group and the nesfatin-1+I/R group than in the sham group(p<0.05);the Bcl-2/Bax ratio was lower than that in the sham group(p<0.05).The expression of protein caspase-3 in brain tissue of nesfatin-1+I/R group was lower than that of I/R group(p<0.05);the ratio of Bcl-2/Bax was higher than that of I/R group(p<0.05).5.The results of Western-Blot assay showed that the expression of caspase-3 in the brain tissue of the I/R group and the nesfatin-1+I/R group was up-regulated compared with the sham group(p<0.05);Bcl-2 The /Bax ratio was lower than the sham group(p<0.05).The expression of caspase-3 in the brain tissue of nesfatin-1+I/R group was lower than that in I/R group(p<0.05);the ratio of Bcl-2/Bax was higher than that in I/R group(p<0.05).Conclusion:1.In rat cerebral ischemia-reperfusion injury,nesfatin-1 preconditioning has protective effects on brain injury and can alleviate brain tissue morphological damage.2.Pretreatment with nesfatin-1 for cerebral ischemia and reperfusion can significantly reduce the volume of cerebral infarction and has a great protective effect on brain injury.3.nesfatin-1 inhibits neuronal apoptosis induced by cerebral ischemia and reperfusion.The possible mechanism of action is down-regulation of caspase-3 and down-regulation of proapoptotic protein Bax.4.Pretreatment with nesfatin-1 for cerebral ischemia-reperfusion injury may protect neuronal apoptosis by up-regulating the expression pathway of anti-apoptotic protein Bcl-2. |
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