| Objective:Epidemiological investigation showed that a large number of cerebrovascular disease among which mainly was ischemia occurred in our country each year.Severe neurological deficits caused by Ischemia which irreversibly resulted in substantial neuronal degeneration and necrosis. At early stage how to promote angiogenesis and local reperfusion to rescue the dying nerve cells in the penumbra was great significance for treatment of ischemic stroke. Vascular endothelial growth factor (VEGF) became the most important factor for directly effect on endothelial cells in angiogenesis. At the same times through the local interaction between cells, Notch signaling pathway influenced the development and formation of blood vessels. Studies had shown that injury by hypoxia and ischemia could increase VEGF expression, activated Notch signaling pathway, and promoted angiogenesis by VEGF regulating Notch signaling pathway. VEGF-Notch signaling pathway played important regulation of development and the formation of blood vessels. However, its specific regulatory mechanisms were not yet fully clarified during which involved the complex relationship among VEGF, VEGFR and Notch signaling pathway. Hypoxia inducible factor-1(HIF-1) was a transcription factor which found in the liver cells and transgenic animals during the erythropoietin(EPO) gene expression study. It could sense specific-hypoxia changes and importantly maintain the oxygen balance of body. HIF-1 participated many pathohpysiologies of angiogenesis, proliferation, apoptosis, energy metabolism, etc through regulating its downstream target genes after ischemia.HIF-1 induced by hypoxia-ischemia comprehensively regulated the expression of VEGF. The results showed that HIF-1-VEGF-Notch signaling pathway was a major regulator of angiogenesis after hypoxia and ischemia.This study mainly observed HIF-1 a, VEGFR2, Notch1 and Hesl in HIF-1-VEGF-Notch signaling pathway in vivo after cerebral ischemia and neovascularization in ischemic region, explored the possibility of HIF-1-VEGF-Notch pathway as the main regulator of angiogenesis mechanism.The results provided a theoretical basis for the future of precise regulation of endogenous brain angiogenesis, tissue repair and achieved functional reconstruction after central nervous system (CNS) injury. It also gave a new treatment idea for ischemic stroke.Methods:1.Sprague-Dawle(SD) rats were randomly divided into sham group and ischemic control group. Local Cerebral ischemia model produced by middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO). Rats were sacrificed at 8 hours, 1 day and 3 days after reperfusion. the brains were removed for inspection.2.Frozen sections of brain samples detected by immunohistochemistry staining the expression of HIF-la in ischemic region and count the number of HIF-1α-positive cells under×400 high power microscope.At the same time, HIF-1αprotein was detected by western-blot assay in ischemic cortex of each samples.3.Frozen sections of brain samples detected by immunohistochemistry staining the expression of HIF-la in ischemic region and count the number of HIF-1α-positive cells under×400 high power microscope in different 5 visions.4.The protein of Notchl and Hesl was detected by western-blot assay in ischemic cortex of each samples, then using image J analyzed its relative expression intensity.5.Frozen sections of brain samples by a double immunohistochemical staining fluorescence detect the expression of Hesl factorⅧto understand the neovascularization in ischemia region.6. Discussion of HIF-1-VEGF-Notch pathway as the main control mechanism of angiogenesis in the ischemic brain.Results1.Sprague-Dawle (SD) rats showed different degrees of the neurological deficits such as contralateral hemiplegia, ptosis, walking around in place,tilt to the opposite side, and raised tail sideways to the opposite side after cerebral ischemia. The brain showed normal red tissue and pale ischemic region by 2,3,5-triphenyl tetrazolium chloride (TTC) staining.2.The positive cells of HIF-1αin the cortex were 2.60±1.52,3.2±1.30,12.60±10.41 after ischemia-reperfusion 8hours, lday and 3days.Compared with the control group,0.40±0.55, the positive cells of HIF-la was significantly increased after 3days. The relative intensity value of HIF-1αprotein detected by western-blot assay were 0.40±0.027,1.76±0.1521 and 38±0.119,whereas the control was 0.48±0.041,at post of ischemia-reperfusion 8hours, lday and 3days.HIF-la protein expression was significantly increased at 1day and 3days.However,the 3days expression declined.3. the positive cells of VEGFR2 in the cortex were 2.40±1.14,3.00±0.7,14.6±9.66 after ischemia-reperfusion 8hours, lday and 3days.Compared with the control group,0.40±0.55, the positive cells of VEGFR2 was significantly increased after 3 days.4. The relative intensity value of Notchl protein detected by western-blot assay were 0.56±0.048,1.10±0.095 and 1.95±0.169,whereas the control was 0.41±0.036,at post of ischemia-reperfusion 8hours, lday and 3days.Notchl protein expression was significantly increased at 1day and 3days which reached a peak at 3 days.The value of Hes1 protein were 0.54±0.049,1.11±0.096 and 1.52±0.131 at 8hours, 1day and 3days.the control was 0.86±0.074. Hesl protein expressions at 8h group was significantly lower than the control. At 1d and 3d the protein increased notably5. the positive cells of Hesl+VIII in the ischemic cortex were9.00±2.24, 24.8±4.44and 36.4±6.27 after ischemia-reperfusion 8hours, lday and 3days. the control group was 14.0±2.35, the positive cells was declined after 8h,after then, increasing to a peak at 3days.Conclusion:1. The detected result suggested that overexpression of HIF-la regulating its downstream taget genes launched a major process of neovascularization which was important significance after ischemia in the cortex.2. Detected the change of VEGFR2,Notchl and Hesl in ischemia cortex, the result suggested that VEGFR2 expression increased and notch signaling pathway activated that effectively promoted angiogenesis under the influence of HIF-1.VEGF-notch signaling pathway had a important regulated role of angiogenesis in ischemic cortex.3. By detecting Hesl+Ⅷfactor in ischemia,the result indicated that HIF-1- VEGF-Notch signaling pathway activated.With the increased expression of HIF-1, VEGF-notch signaling pathway enhanced the proliferation of endothelial cells and effectively promoted angiogenesis. HIF-1-VEGF-Notch signaling pathway became a major regulatory way in angiogenesis after ischemia. |
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