| It has been believed that mature neurons in the central nervous system are terminally differentiated, and cannot proliferate. Thus, it seems that mature neurons can hardly self-repair when injured. However, recent several studies have successfully cultured neural stem cells(NSCs) from adult animal brain and showed they have proliferative potential. These findings bring the new hope for the repair of injured central nervous system. Unfortunately, to data most of present methods used to promote the proliferation of NSCs are hardly available in clinical treatments because of weak clinical operability. Therefore, finding a drug which can promote NSCs proliferation and oriented differentiation is urgent for solving this problem. GSRd is one of the main ingredients of Panax ginseng and Panax notoginseng. Our previous clinical and basic studies have shown an overt neuroprotection of GSRd. For instance, it was safe and effective to treat acute ischemic stroke patient, reduced the infarct volume of injured rats, decreased neuronal cell death, and ameliorated the functional recovery of cerebral ischemic animals. However, the effects of GSRd on the neurogenesis of NSCs have not been specifically addressed. In this study we aimed to investigate the effects and the underlying mechanisms of GSRd on the proliferation of NSCs in vivo and in vitro.Part 1 Effects of GSRd on the neurogenesis of hippocampal dentate gyrus in adult ratsObjective: To explore the effects of GSRd on the proliferation and differentiation of NSCs in hippocampal dentate gyrus. Methods:(1) By continuously intraperitoneally injecting 30 mg/kg of GSRd to adult rats once a day for 1 week, the rats were executed at 7 day and 14 day after the administration. The effects of GSRd on the hippocampus NSCs proliferation were observed by Brd U and DCX staining.(2) The effects of GSRd on the hippocampal NSC differentiation were examined by Brd U/Neu N and DCX/GFAP double fluorescence staining at 34 day after the administration. Results:(1) GSRd significantly increased the numbers of Brd U+ and DCX+ cells in the hippocampal dentate gyrus, compared with the control group at 7 day(P<0.05). However, 14 day after the administration of GSRd, there was no difference in the number of Brd U+ and DCX+ cells between these two groups.(2) GSRd significantly increased the numbers of Brd U+ and DCX+ cells in the hippocampal dentate gyrus(P<0.05) but did not affect the ratio of Neu N+/Brd U+ double-labeled cells to the total number of Brd U+ cells Additionally, no GFAP +/Brd U+ double-labeled cells were observed in both groups. Conclusion: GSRd significantly increased the number of NSCs and new-born neurons in the hippocampal dentate gyrus, but did not affect their differentiation.Part 2 Effects of GSRd on the proliferation and differentiation of NSCs in vitroObjective: To explore the effects of GSRd on the proliferation and differentiation of NSCs in vitro. Methods:(1) NSCs were isolated and cultured, and then were identified by immunocytochemistry.(2) After treated NSCs with GSRd at different doses, the changes in diameter and the numbers of neurosphere were measured. Brd U immunocytochemistry was used to examine NSC proliferation.(3) Tuj-1, GFAP and CNPase immunocytochemistry were used to examine the differentiation of NSCs. Results:(1) The cultured cells were Nestin and Ki67 positive and could differentiated into Tuj-1+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes.(2) The diameter and number of neurosphere in GSRd group were much more than the control group(P<0.05). Similarly, the number of Brd U+ cells in GSRd group was more than the control group(P<0.05).(3) There were no difference in the percentages of Tuj-1+, GFAP+ and CNPase+ cells between these two groups. Conclusion: GSRd significantly promoted the proliferation of NSCs in vitro, but failed to exert its effect on NSC differentiation.Part 3 Possible mechanisms underlying GSRd-induced proliferation of NSCsObjective: To explore the mechanism of the promotive effect of GSRd on NSC proliferation. Methods:(1) The protein levels of Akt, phospho-Akt(p-Akt), ERK, phospho-ERK(p-ERK), PKC, and phospho-PKC(p-PKC) were examined by Western blot in NSCs after GSRd administration.(2) The protein levels of Akt、p-Akt、ERK、p-ERK、PKC and p-PKC were examined by western blot in GSRd-treated NSCs after intervention of PI3K/Akt inhibitor LY294002 or MAPK/ERK1/2 inhibitor PD98059.(3) The number of Brd U+ cells in GSRd-treated NSCs was examined by immunocytochemistry after intervention of LY294002 and PD98059. Results:(1) GSRd significantly elevated the protein expression levels of p-Akt、p-ERK(P<0.05), but not of Akt、ERK、PKC and p-PKC.(2) The protein levels of p-Akt and p-ERK decreased in GSRd-treated NSCs after the intervention of LY294002 and PD98059(P<0.05).(3) The promotive effects of GSRd on NSC proliferation were attenuated when treated with LY294002 and PD98059(P<0.05). Conclusion: PI3K/Akt and MAPK/ERK1/2 signaling pathways may be involved in GSRd-induced proliferation of NSCs. |
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