Study On Protective Effect And Mechanism Of Ginkgolide B On Embryonic Rat Midbrain Neurons

Objective: In order to provide the benificial use of GB in the treatment of illness caused by midbrain damage, We investigated the protective effect and the mechanism of Ginkgolide B on embryonic rat midbrain neurons in vitro. Methods: We used midbrain neurons cell suspension from E-14 day old Wistar rats and kainic acid induced midbrain neurons injury as the study models.The NGF was performed as a comparision substances with GB. The cell survival rate was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT) assays. The NSE positive neurons in the cultured cells were detected by using SP histochemical stain. The neuron morphological changes made by Leica were detected. The activities of SOD, the contents of MDA, NO and the protein in the cultrued cells were determined for exploring the mechanisms of GB on the embryonic rate mesencephalic cells.The methods above were prepared to investigate the effects of protection and the mechanisms of GB on mesencephalic cells cultured in vitro.Results:1. The effect of GB on the growth of midbrain nerve cells1.1 The survival ratio of midbrain nerve cells: Compared with the control group, the ratio of cell survival of NGF and GB groups was increased significantly when the midbrain cells were cultrued in the third(P<0.05) or fourth day(P<0.01), and there was no difference between NGF and GB group. The cell survival ratio of KA group was lower obviously than that of control group in the second day(P<0.05). The cells, exposed to KA, showed characteristic change of damage, which could be relieved by the treatment of NGF and GB, and GB showed the dose-dependent manner (P<0.05).1.2 Colony differentiation ratio of embryonic rate midbrain nerve cells Compared with the control group(100%), the colony diffrentiation ratio of NGF(133%) and GB(134%) group were increasing obviously(P<0.01), and KA can inhibit the colony diffrentiation of embryonic rate midbrain cells significantly(29.4%, P<0.01). NGF and GB can partly prevent the embryonic rate midbrain cells from damage by KA(86.5%, 82.1%, respectively, P<0.01, NGF and GB group vs KA group).2 The effect of GB on NSE expression of midbrain nerve cells2.1 The quantity of NSE cells: Compared with the control group, the number of NSE cells in NGF and GB group was increasing significantly, and the number of NSE cells in KA group was decreasing obviously. In addition, the number of NSE cells in GB+KA and NGF+KA groups was higher than that of KA group(P<0.01).2.2 The area of NSE cells: The cell average area in NGF and GB groups were 1207μm2 and 1167.7μm2 respectively, which was larger than that of control group(1148.6μm2, p<0.05), and there was no difference between NGF and GB group. In contrast, the cell average area in the KA group was 132.2μm2, which was smaller sharply than that of control group(P<0.01). Furthermore, the average area of NSE cells in GB+KA and NGF+KA group were smaller than that of control group, but were larger than that of KA group(P<0.01).2.3 The number of protuberance of each NSE cell: The average number of protuberance of each NSE cell in NGF and GB group were 3.92A/cell and 3.96 A/cell respectively, which was higher than that of control group(P<0.01).Similarly, the average number of protuberance of each NSE cell in GB+KA and NGF+KA group were 2.05 A/cell and 2.38 A/cell respectively, which was higher than that of KA group(1.03, P<0.01).2.4 The longest length of NSE cell processes: The average longest length of each NSE cell processes in GB and NGF group were 91.28μm and 88.54μm respectively, which was longer than that of control group(79.44μm, P<0.01). Further, the average longest length of each NSE cell processes in GB+KA and NGF+KA group were 62.46μm and 62.35μm respectively, which was longer than that of KA group(P<0.01).3 The effect of GB on the contents of NO, MDA and relative protien and the activity of SOD in the cultrued cells of embryonic rate midbrain: GB(25-100 mg/L) can prevent the nerve cells from damage by KA with its concentration increasing. The contents of No and MDA in nerve cells in GB(50 mg/L) group was similar to that of control group in the cells cultrued to 10th day. However, the activity of SOD and the content of relative protein in the cells were higher than that of control group. The contents of cellular NO and MDA in the NGF(50mg/L)+ KA(100μmol/L) and GB(50mg/L)+KA(100μmol/L) groups were lower than that of KA(100μmol/L )group, but the content of cellular protein and the activity of SOD were higher than that of KA(100μmol/L) group significantly.Conclusion: GB can promote the development of embryonic rate midbrain neuronic cells and prevent the cells from damage by KA, which may relate with the NSE expression in the glucose metabolism process accelerated by GB. It also relate with the course of oxygen free radicals balance reinstated by GB after the midbrain cells were damaged. The results suggested that GB would be benificial for the treatment of illness caused by midbrain damage.