| Background The restoration in ischemic zone need new blood vessels organization provides nutrient substance , and maintaining its normal tissue metabolism. As soon as the recovery of blood supply in ischemic penumbra, the dying neurons can be saved. That will be helpful for neural functions to restore. It is the key of cerebral ischemia disease therapeutics. Super early fibrinolytic therapy is limited in the clinical application due to the time window and its success rate. The Treatment Effectiveness of extension vascular, anticoagulation, antiplatelet and so on is poor too. So, how to mobilize endogenous angiogenesis to promote capillary formation and collateral circulation reconstruction in ischemic region has become a hotspot of the present research. Through acupuncture, which from the surface points, stimulating the endogenous protection mechanism of the body is an effective method of non-drug treatment way after cerebral ischemia.The recent studies found that endothelial progenitor cells (EPCs) is closely related to the vascular regeneration. after cerebral ischemia, the cerebral ischemic region release some cytokines, which can activate the signal transduction pathways related in a series of cascade to mobilizes bone marrow EPCs homing. PI3K/AKT is the typical signal transduction pathways related to the EPCs proliferation, migration and homing. Stromal cells derived factor-1 alpha (SDF-1α) combining with CXCR4 can activate PI3K/AKT signal transduction pathways. Previous studies of our experimental group showed that Electroacupuncture(EA) can up-regulate SDF-1αexpression and promote the blood regeneration in cerebral ischemic region. But the mechanism of EA promoting EPCs homing are still unclear. This experiment is starting from the problem discussed.This subject focuses on the changes of the expression of AC133+ EPCs and the activation of PI3K/AKT signal transduction pathways in rat brain under intervention of electroacupuncture and LY294002 (PI3K inhibitor) after focal cerebral ischemia/reperfusion. To investigate the mechanism of EA on promoting revascularization in the rat brain of focal cerebral ischemia/reperfusion mediated by PI3K/AKT signaling pathway. To provide experimental basis For EA treatment of ischemic cerebrovascular disease.Objective1. To investigate the changes of the expression of AC133 in rat brain under intervention of electroacupuncture after focal cerebral ischemia/reperfusion and to ensure whether electroacupuncture can promote the EPCs homing.2. To study the role of PI3K/AKT signal transduction pathways on revascularization after focal cerebral ischemia/reperfusion.3. To investigate the mechanism of EA on promoting revascularization in the rat brain of focal cerebral ischemia/reperfusion mediated by PI3K/AKT signaling pathway.Methods The 80 male SD(Sprague-Dawley) rats received filament occlusion of the right middle cerebral artery for 2 hours. Rats were randomly divided into control group( NC group ) , model group (I/R group ) , EA group ( I/RE group) , EA+ LY294002 group( I/RA group ). According to accept reperfusion 1d, 3d, 7d after 2h ischemia, the model group and EA group were divided into three subgroups. Each subgroup has 10 rats. After 1h of the reperfusion, EA was given at bilateral"Hegu"point (LI 4) in the EA group and EA+ LY294002 group. These rats were treated with continuous stimulation for 15min each day, up to 7 days. I/RA group selects the phase point of reperfusion 3d to observation. The rats of I/RA group was injected LY294002 to lateral ventricles after 2h ischemia and before EA 1h. I/RN group and I/RA group has 10 rats.To evaluate the nerve function defects of focal cerebral ischemia/reperfusion by neurological symptom scores. HE staining is used to observe pathology diversity of brain tissue. Immunohistochemical method was used to detect the expression of p-AKT1 and AC133 protein in the ischemic penumbra. Reverse transcription- polymerase chain reaction(RT-PCR) was used to detect the expression of AC133 mRNA. Western blot was employed to detect the expression of p-AKT1 and AKT1 protein.Results 1. Neurological symptom scoreAt the time points of reperfusion 2h, 1d, 3d, the difference of neurological symptom score between I/RE group and I/R group was not statistically significant(p>0.05). As ischemia-reperfusion prolonged, neurological symptom score was gradually reduced and the neural function of rats have somewhat restored. At the time points of reperfusion 7d, I/RE group and I/R group had a statistically significant difference (p<0.05).2. The expression of AC133 mRNANC group has a small amount of AC133 mRNA expression on the right cerebral cortex. In I/R group, AC133 mRNA expression has a single peak-like increase, compared with NC group, increased strikingly at 3d (P< 0.05), reached a peak value at 7d point (P<0.01). I/RE group compared with I/R group, At the time points of reperfusion 3d, 7d, AC133 mRNA expression of I/RE group is higher than I/R group, the difference between them was statistically significant (p<0.05). In I/RA group, AC133 mRNA expression was obviously lower than I/RE group(p<0.05), but there was no difference between I/RA group and NC group. 3. AC133 protein immunohistochemical result analysisIn NC group, the expression of AC133 protein was negative. I/R group: At the time points of reperfusion 3d, Ischemic penumbra in the right hemisphere showed a few of brown cells that means AC133 protein expression was positive, but the other time points were negative. I/RE group: At the time points of reperfusion 3d, 7d, AC133 protein expression was increased. Compared with I/R group, the difference was statistically significant (P<0.05). I/RA group: Because of LY294002, AC133 protein expression was negative.4. P-AKT1 protein immunohistochemical result analysisNC group and I/RA group p-AKT1 protein expression was negative; I/R group: p-AKT1 protein mainly expressed at ischemic penumbra in the right hemisphere of rats. Compared with NC group, p-AKT1 protein expression reached a peak value at 1d point(P<0.01), then decreased gradually at 3d point(P<0.01), but still higher than the NC group at 7d point (P<0.05). I/RE group have a large number of brown cells in ischemia penumbra area, and cell shading obviously deepened. Compared with I/R group ,the difference between them was statistically significant at the time points of reperfusion 3d, 7d(P<0.01).5. Western blot to detect the expression of p-AKT1 proteinp-AKT1 protein as a 60KD curvilinear is consistent with its actual molecular size.NC group has a few p-AKT1 protein expression. In I/R group: p-AKT1 protein expression was increased, reached a peak value at 1d point(P<0.01), then began to decline at 3d point(P<0.01), but still higher than the NC group at 7d point (P<0.05). I/RE group: p-AKT1 protein expression was increased obviously, reached a peak value at 3d point, then decreased gradually at 7d point. At the time points of reperfusion 3d, 7d, I/RE group compared with I/R group, the difference between them was statistically significant (P<0.01). In I/RA group, p-AKT1 protein expression was obviously lower than I/RE group(p<0.01), but there was no difference between I/RA group and NC group. But AKT1 protein expression between groups has no significant difference (P > 0.05).Conclusion1. EA can effectively improve the neurologic deficits and promote the nerve functional recovery of the focal cerebral ischemia/reperfusion rats by promoting the endogenous angiogenesis.2. EA may be strengthened bone marrow-derived EPCs homing to the ischemic zone that made its expression increase.3. EA could promote revascularization in the rat brain of focal cerebral ischemia/reperfusion mediated by activating PI3K/AKT signaling pathway.4. EA can promote bone marrow-derived EPCs homing to the ischemic zone by activating PI3K/AKT signaling pathway.
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