| Objective: To explore the neuroprotective mechanism of rapamycin(RAP)by regulating autophagy and Wnt/β-catenin signaling pathway after cerebral ischemia reperfusion injury(CIRI)in rats,with a view to clinical treatment ischemic stroke provides a new target.Methods: 180 clean,healthy male(Sprague Dawley,SD)rats were selected and randomly divided into sham operation(sham)group,middle cerebral artery occlusion(MCAO)group and rapamycin pretreatment(RAP)group,60 animals in each group.In the RAP group,5μL of RAP with a concentration of 1mmol/m L was injected into the lateral ventricle after 1h of MCAO;the sham group and MCAO group were given the same dose of normal saline in the lateral ventricle.Then perform the following tests:1.Morris water maze and new object recognition experiment.2.Neurological deficit score.3.Magnetic resonance(MRI)imaging to observe cerebral ischemia.4.2,3,5-Triphenyltetrazolium chloride(TTC)staining.5.Brain tissue water content detection.6.HE and Nissl staining.7.Malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX)kits for determination of MDA content,SOD and GSH-PX Enzyme activity.8.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-1β(IL-1β)and interleukin-8(IL-8),factors related to inflammation in hippocampus.9.Dihydroethidium(DHE)fluorescence staining to detect reactive oxygen species(ROS)levels.10.TUNEL detects cell apoptosis in the CA3 area of the hippocampus.11.TUNEL and Neu N co-localization and TUNEL and GFAP co-localization detect neuron and astrocyte apoptosis respectively.12.Immuno-histochemical staining(IHC)staining to detect the expression of Neu N,Bcl-2,Bax,LC3 II/I,Beclin-1,GSK-3β and β-catenin positive cells in hippocampal CA3 area.13.Immunohistochemical fluorescence(Immunohistochemical fluorescence,IF)single-label staining method to detect the expression of Neu N,GFAP,LC3 II/I,Beclin-1,β-catenin and GSK-3β positive cells in hippocampal CA3 area.14.Immunofluorescence double-label staining method to detect the changes of GSK-3β and LC3 II/I,LC3II/I and Neu N in hippocampal CA3 area.15.Laser confocal observation of Neu N and TUNEL,GSK-3β and Neu N,LC3II/I and Neu N,GSK-3β and LC3 II/I co-localized expression.16.q RT-PCR detection of hippocampal LC3 II/I,Beclin-1,β-catenin,GSK-3β m RNA content in each group.17.Western blot(WB)was used to detect the expression of Beclin-1,LC3 II/I,P62,Bcl-2,Bax,GSK-3β,and β-catenin in rat hippocampus.18.Transmission electron microscope(TEM)observation of the ultrastructure changes of hippocampal cells in each group.Results: 1.Morris water maze and new object recognition results: Compared with the MCAO group,rats in the RAP group have a lower escape latency,an increase in the number of crossing platforms,a longer stay in the target area,a shorter swimming distance,and a clear direction of the movement track.Compared with the MCAO group,the rats in the RAP group had significantly longer time to explore new objects and familiar objects.At the same time,the rats in the RAP group had significantly less activity than the MCAO group,and the relative recognition index was higher than that in the MCAO group.2.Results of neurological function assessment: The neurological deficit score of rats in the RAP group was significantly lower than that in the MCAO group.3.MRI imaging test results: Compared with the sham group,the MCAO group showed large patches of high signal on the right cerebral hemisphere on T2 WI,low signal on T1 WI,high signal on DWI,reduced ADC value,and reduced FA value.After 3D-ASL the processed images showed that the CBF of the right middle cerebral artery blood supply area was reduced;compared with the MCAO group,the relative area of the cerebral hemisphere patchy hyperintensity and DWI abnormal hyperintensity area on the T2 WI obstructed side of the RAP group and the relative area of the PWI perfusion defect area were smaller than those in the MCAO group,r ADC the value and r CBF value are larger than MCAO.4.TTC staining results: the cerebral infarction volume of rats in the RAP group was significantly lower than that in the MCAO group.5.Brain water content results: The brain water content of the RAP group was significantly lower than that of the MCAO group.6.HE staining results: Compared with the sham group,the cytoplasm of the hippocampal CA3 area of the MCAO group was lightly stained and the nerve cell necrosis was obvious;compared with the MCAO group,the nucleus of the hippocampal CA3 area of the RAP group was clearer and the nerve cell damage was reduced.7.Nissl staining results: Compared with the sham group,the neurons in the hippocampal CA3 area of the MCAO group showed vacuolar degeneration,pyknosis of the nucleus,dissolution or disappearance of Nissl bodies,and severe neuronal damage;compared with the MCAO group,the hippocampus in the RAP group the nerve cells in CA3 area are arranged more neatly,the number of Nissl bodies increases,and the nerve cell damage is significantly reduced.8.Oxidative stress test results: Compared with the sham group,the activities of SOD and GSH-Px in the hippocampus of the MCAO group were significantly reduced,while the content of MDA was significantly increased;compared with the MCAO group,the SOD and GSH-Px enzymes in the hippocampus of the RAP group were significantly increased the activities are significantly increased,while the MDA content is significantly reduced.9.ELISA test results: RAP group significantly reduced the expression of inflammatory factors IL-1β and IL-8 in the hippocampus of rats.10.DHE fluorescent staining results: RAP group significantly lower ROS level than MCAO group.11.TUNEL test results: Apoptotic cells in hippocampal CA3 area of RAP group were significantly lower than that of MCAO group.12.TUNEL and neuron marker(Neu N)co-localization test results: Compared with the sham group,the apoptotic cells in the ischemic penumbra of the hippocampal CA3 area of the MCAO group increased significantly,and the co-localization of TUNEL and Neu N increased significantly;Compared with the MCAO group,the apoptotic cells in the ischemic penumbra of the hippocampal CA3 area of the RAP group were significantly reduced,and the apoptotic cells in the co-localized neurons of TUNEL and Neu N were significantly reduced.13.TUNEL and astroglial marker(GFAP)co-localization test results: Compared with the sham group,the ischemic penumbra of hippocampus CA3 area of the MCAO group showed a significant increase in astroglial apoptosis,and TUNEL and GFAP co-localized compared with the MCAO group,astrocytes in the ischemic penumbra of the hippocampal CA3 area of the RAP group were significantly reduced,and TUNEL and GFAP co-localized and co-localized astrocytes significantly decreased.14.IHC test results: Compared with the sham group,the expressions of Bcl-2 and Neu N in the hippocampal CA3 area of the MCAO group decreased,and the expressions of Bax,β-catenin,GSK-3β,LC3 II/I and Beclin-1 increased significantly;Compared with the MCAO group,the expressions of Bcl-2 and Neu N in the hippocampal CA3 area of the RAP group were significantly increased,and the expressions of Bax,β-catenin,GSK-3β,LC3 II/I and Beclin-1 were significantly reduced.15.IF test results: Compared with the sham group,the expression of Neu N positive cells in the hippocampus CA3 area of rats in the MCAO group after CIRI was significantly reduced,while GFAP,β-catenin,GSK-3β,Beclin-1 and LC3 II/I positive cells Compared with the MCAO group,the expression of Neu N positive cells in the hippocampal CA3 area of the RAP group increased significantly,while the expression of GFAP,β-catenin,GSK-3β,Beclin-1 and LC3 II/I positive cells decreased significantly.16.Immunofluorescence double-labeled detection of LC3II/I and GSK-3β and LC3II/I and Neu N colocalization results: Compared with the sham group,the MCAO group LC3II/I and GSK-3β and LC3II/I and Neu N colocalized cells significantly increased;Compared with MCAO group,LC3II/I and GSK-3β and LC3II/I and Neu N co-localized cells in RAP group were significantly reduced.17.LC3II/I and GSK-3β,LC3II/I and Neu N,GSK-3β and Neu N,Neu N and TUNEL laser confocal observation results: Compared with the sham group,the MCAO group has significantly more co-localized cells,compared with the MCAO group the co-localized cells increased significantly,in the RAP group.18.RT-PCR detection results: Compared with the sham group,the m RNA expression levels of hippocampal β-catenin,GSK-3β,LC3 II/I and Beclin-1 in the CIRI group were significantly up-regulated;compared with the MCAO group,the RAP group rat hippocampus β-catenin,GSK-3β,LC3 II/I and Beclin-1 were all significantly down-regulated.19.Western blot detection results: Compared with the sham group,the hippocampal β-catenin,GSK-3β,LC3 II/I and Beclin-1 protein expression in the MCAO group increased significantly;compared with the MCAO group,the RAP group rats hippocampal β-catenin,GSK-3β,LC3 II/I and Beclin-1 protein expressions were all significantly reduced.20.TEM observation results: Compared with the sham group,neurons in the hippocampus of the MCAO group were edema and decreased,the mitochondrial cristae disappeared and swelled,and even vacuolated,apoptotic bodies appeared,and a large number of autophagosomes were seen in autophagy lysosome accumulation.After RAP treatment,the structure of mitochondria is relatively complete,with a small amount of autophagolysosomes and autophagosomes.Conclusion: 1.RAP significantly improves the neurological deficits of MCAO rats;reduces the area of cerebral infarction and cerebral edema;MRI shows that it improves the cerebral blood flow of ischemic tissue;it improves the ability of spatial learning and memory and new object recognition in rats.2.RAP significantly improves neuron necrosis caused by CIRI,reduces oxidative stress and inflammation,inhibits astrocyte activation,and promotes the repair of damaged brain tissue.3.Both autophagy and Wnt/β-catenin signaling pathways have a key shared protein GSK-3β;RAP may negatively regulate the expression of Wnt/β-catenin signaling pathway and autophagy shared protein GSK-3β to exert neuroprotection on CIRI in SD rats effect. |
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