| Cerebral ischemia/reperfusion injury is the main cause of ischemic cerebrovascular diseasesto humans, especially for the elderly persons who would suffer more risks. Therefore, it is extremely urgent to find out an effective means of treatment. The most common used ways to cure this disease are mainly about thrombolytic and neuroprotective therapy, which aimed at restoring neurological defects with little effects. Thankfully, it provides new hope with the observation of the phenomenon on neurogenesis for curing the cerebral ischemic diseases. Recent studies have shown that Chinese medicines have excellent prospects in promoting neurogenesis. This paper intends to investigate the effect of ginsenoside Rd on promoting neurogenesis after cerebral ischemia/reperfusion and its potential mechanisms via the PI3K/Akt pathway.Parti:We established the classic middle cerebral artery occlusion model to result in the right brain side of SD rats with focal brain ischemia. Sham group, model group and three different concentration gradients of ginsenoside Rd groups were set in this part. The rats of model group and three doses of ginsenoside Rd groups were subjected to brain ischemia for 90 minutes followed by reperfusion for 24 hours. The influence of different concentrations of the drug to nerve function in rats was observed by using a 0-4 neurobehavioral scores. The drug’s effective dose was measured by calculating the area of the cerebral infarction. We also used the morris water maze to test the improvement on learning and memory abilities by the Rd and used western blot to detect the Akt protein expression by different concentrations of Rd. Our experimental results showed that 5mg/kg dose of ginsenoside Rd could significantly reduce the neurobehavioral scores and the cerebral infarct size, improve the cognitive and memory capacity, together with an increasing in the ratio of p-Akt/Akt.Part2:The way to establish animals’model was the same with the first part. The sham group, model group, model+ginsenoside Rd (5mg/kg) group and the model+ginsenoside Rd (5mg/kg)+LY294002 group were set in this part. By using the methods of neurobehavioral scores and western blot, the impact on the nerve function and the expression of Akt protein after cerebral ischemia/reperfusion injury in rats with the treatment of PI3K inhibitor were observed, respectively. Furthermore, we used BrdU/DCX and Nestin/GFAP double immunofluorescence to survey the effect of ginsenoside Rd on promoting neurogenesis and the blocking of PI3K pathway. Ours experimental results showed that the PI3K. inhibitor would block the neuroprotective effects of ginsenoside Rd. Otherwise, the 5mg/kg Rd could increase the number of BrdU/DCX and Nestin/GFAP positive cells by 7 days after reperfusion. whereas, this effect would by inhibited by PI3K blocker.Part3:The oxygen glucose deprived procedure was put on the PC 12 cells for four hours followed with reoxygenation for six hours. Respectively, the 25,50 and 100μmol/l of ginsenoside Rd was intervented in cells. The enzyme-linked immunosorbent assay was used to detect the secretion of VEGF and BDNF in supernatants. The western blot assay was used to test the expression of Akt and ERK protein as well as the blocking. The flow cytometry was used to inspect the apoptosis of cells as well as the blocking. Our experimental results showed that the secretion of VEGF and BDNF significantly increased after ginsenoside Rd treatment. In addition, the phosphorylation of Akt and ERK protein levels could also be significantly increased by Rd with the increasing of the cell survival. However, all these effects would be blocked by a PI3K inhibitor.This project shows that ginsenoside Rd can protect brain tissues after ischemia/reperfusion injury with the restoration of nerve function. Moreover, it can further promote neurogenesis and the proliferation, differentiation and migration of neural stem cells. The effects of ginsenoside Rd may work via the upregulation of VEGF and BDNF, and partly by the activation of PI3K/Akt and ERK 1/2 signaling pathways. These findings provide instructive purpose on curing the cerebral ischemia/reperfusion diseases, as well as provide a basis for the further continued study of ginsenoside Rd. |
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