Effect And Mechanism Of Three Active Ingredients On Regulating Astrocytes After Stroke

ObjectiveNeural stem cell niche can provide a microenvironment for damaged neural stem cells in the brain. The nest mainly has neural stem cells, astrocytes and brain microvascular endothelial cells. Astrocytes may play a role to some extent in the repair of the nervous system, they activate the process of migration of neural stem cells in the brain injury, serving as a guide through this way to repair damaged brain tissue. We study the role of astrocytes during brain injury, especially mechanism of action during cerebral ischemia. This study use ginsenosides、astragaloside and Ophiopogonin D respectively interve astrocytes after cerebral ischemia reperfusion, study the role of active ingredients of traditional Chinese medicine on proliferation of astrocytes.Discussion the fuction and mechanism of ginsenosides、astragaloside and Ophiopogonin D after stroke on astrocytes by regulating HIF-la-VEGF, to provide foundation for treating central nervous system diseases used ginseng、astragalus、Ophiopogon and its active ingredient.Methods1 Astrocytes were isolated from the newborn 1-3d SD rats cerebral cortex. We used methods as differential adhesion and multiple passages to purify cells, GFAP labeled and immunofluorescence identified the cells. GFAP is astrocytes specific marker, DAPI marked nuclei can be non-specific, positive cells were more than 95%. So the method described above can achieve the purpose to purify primary cultured astrocytes. By building OGD/Reperfusion (OGD/R) model simulated cerebral ischemia and reperfusion injury, established four time points of hypoxic, the same screening OGD best time point using MTS assay.2 The astrocytes made into single cell suspension, add ginsenosides、astragaloside and ophiopogonin D to single cell suspension of astrocytes, then set up eight different concentration points using MTS assay OD values to calculate the concentration of different eight points on proliferation of astrocytes, we get the three drugs most good concentrations were added to astrocytes single cell suspension respectively, set up eight time points and obtained the best time of the three drugs to promote proliferation by MTS.3 The in vitro cultured astrocytes divided into normal group, model group namely ischemia-reperfusion group and treatment group (administration of concentration after reperfusion), the normal cells cultured in conventional incubator, the model group and the treatment group were hypoxia cultured for 4h in three gas incubator, then reoxygenation cultured 2h、4h、6h, detect the expression of astrocyte-specific marker of GFAP by immunofluorescence, calculate the number of positive cells, optical density and surface density.4 The application of Western blot analysis the content of HIF-1α protein about normal group, model group and treatment group; Detection the content of vascular endothelial growth factor (VEGF), which in the cell culture supernatanteach in each group after each time point by ELISA.Results1 The trend of its OD value that astrocytes induced by hypoxia rose at first then declined, the optimal duration of action point 4h.2 The optimal dosing concentration of ginsenosides、astragaloside and ophiopogonin D which promote astrocytes proliferation was 12.5μg/ml、0.05μg/ml and 0.005μg/ml; the best effect time of the three drugs was 72h、48h and 72h.3 Compared with the normal group, hypoxia 4h then reoxygenation 2h、4h、6h, the number of positive cells、optical density and surface density in model group were decreased, the difference was statistically significant (P<0.05);compared with the model group, the number of positive cells、optical density and surface density in administration group of ginsenosides、astragaloside and ophiopogonin D were increased significantly, the difference was statistically significant (P<0.05),but less than the normal group, indicating that ginsenosides、astragaloside and ophiopogonin D can promote astrocyte proliferation which is injury in cerebral ischemia-reperfusion.4 It was found that the expression of HIF-la was fewer in normoxic conditions, the expression increased after hypoxia, compared with the normal group, ischemia-reperfusion model group has the difference on statistically significant (P<0.05);compared with the model group, HIF-1α protein increased in the treatment group, the difference was statistically significant (P<0.05), reoxygenation 2h、4h、6h time points showed upward trend. The expression of VEGF protein in cell culture supernatants of each group was the same as HIF-la protein, where less VEGF protein expression in normal condition, later increased expression under hypoxia condition. Compared with the normal group, the model group was statistically significant (P<0.05);compared with the model group, the content of VEGF protein increased in the treatment group, the difference was statistically significant (P<0.05). Indicating that HIF-la expression in astrocytes increased after hypoxia, induction of VEGF expression increased in downstream, ginsenosides、astragaloside、ophiopogonin D promotes the expression of HIF-la and VEGF protein in astrocytes after ischemia-reperfusion injury.ConclusionGinsenosides、astragaloside and ophiopogonin D increased the content of VEGF and HIF-1α protein in the cell culture supernatant after hypoxic, may also increase the number of positive cells, optical density and surface density after ischemia and reperfusion, suggesting that the three active ingredients have a protective effect on astrocytes that caused by ischemia-reperfusion injury. Ginsenosides、astragaloside and ophiopogonin D can start expression of downstream target genes VEGF by regulating the form of paracrine and autocrine on astrocytes, and promote the proliferation of astrocytes to repair brain damage.