Explore Rhizoma Ligustici Chuanxiong Guiding Action In Buyang Huanwu Decoction From The Angle Of Neurogenesis

Objective:Cerebral ischemia/reperfusion model of MCAO was established to investigate the effect of R、NR、AC on the recovery of neurological function and NSCs proliferation, migration, differentiation; NSCs and BMECs were isolated from new born rats, and co-cultivated by a new type system, observe the the Chuanxiong active component TMP on angiogenesis and NSCs proliferation, migration in the co-cultivated system. To reveal molecular basis of NSCs by R promoting, from a new perspective to clarify the guiding action of R and provide provide experimental basis for the essence of guiding theory.Methods:Cerebral ischemia/reperfusion model of MCAO was established in rats using the suture method, successful modeling were divided ischemia model group, AC group, R group, NR group randomly. All groups were administered orally24h after ischemia once a day for7and21days. Another16rats just don’t block the artery rats set to Sham. Neurological fucation was evaluated with neurologic deficit after1W,2W,3W. And in4th and18th day inject BrdU by intraperitoneal,50mg-kg-1,3d. The cerebral ischemia area was determined by TTC staining.BrdU+cells, SDF-1/CXCR4, Reelin, VEGF proteins were examined by immunohistochemistry, BrdU+/DCX+, BrdU+/Map-2+, BrdU+/GFAP+cells were measured by imunofluorescence. western blot and ELISA analyzed the expressions of SDF-1, CXCR4, BDNF, VEGF, FGF proteins.cell proliferative activity are detected by the incubation of MTS and BrdU incorporation, to observe the effect of TMP on the proliferation of NSCs and BMECs.The influence of TMP on the migration of NSCsand BMECs in the co-cultivated system are observed through the Transwell inserts; the role of TMP on BMECs tube formation ability are observed through the Matrixgel enveloped96well board; observe BMECs tube formation ability in the co-cultivated system by Matrixgel enveloped96transwell board and TMP intervention.Results:1. MCAO model and efficacy evaluationModel rats showed severe neurological disfuction compared with Sham group at1W,3W after ischemia. Rats receiving AC, NR, R had a more rapid recovery of neurologic deficit, and AC could significantly reduce neurological behavioral score and cerebral ischemia areas(P<0.05or P<0.01).2. AC, NR, R on NSCs proliferation, migration, differentiation of MCAO ratsModel rats BrdU+, BrdU+/DCX+, BrdU+/Map-2+, BrdU+/GFAP+cells enhanced compared with Sham group at1W,3W after ischemia. positive cells in drug intervention groups was more than in Model. The cells in AC group were obviously more than that other two groups(p<0.05, p<0.01).3.factor of NSCs in MCAO rats AC, NR, R on migrationImmunohistochemistry, western blot results showed that drug intervention groups can improve SDF-1expression at1W after ischemia.and AC group was significantly increased (P<0.05, P<0.01). Similarly, the expression of its specific receptor CXCR4also showed similar trends. SDF-1expression at a higher level at3W after ischemia, and its specific receptor CXCR4expression is also maintained at a high level. Immunohistochemistry, western blot results showed that the treatment groups can improve the expression of VEGF, Reelin in MCAO rats at1W,3W after ischemia, and NR and AC has a significant difference(P<0.05, P<0.01). ELISA results showed that only AC group can improve the expression of BDNF significantly.4.Rhizoma Ligustici Chuanxiong active component TMP on angiogenesis and NSCs proliferation, migration in the co-cultivated system of NSCs/BMECs MTS and BrdU incorporation showed that NSCs, BMECs proliferated significantly When the TMP concentration increased2-50ug·ml-1(P <0.05,when the TMP was increased to more than200ug·ml-1no significant effect on the proliferation.Compared with the Control group, VEGF induced group can significantly increase BMECs tube formation in the Matrigel, with the increase of concentration,TMP can dose-dependently promote BMECs tube formation.(P<0.01). and TMP10-50ug-ml-1has a significant difference(P<0.05, P<0.01).Compared VEGF-induced the BMECs tube formation, Number of branch points increased significantly in the co-cultivated system(P<0.05, P<0.01)with the increase of concentration, tube formation capacity of BMECs enhanced.Transwell results showed that the lower chamber BMECs can promote NSCs in upper chamber migration, and NSCs can also promote the BMECs downward migration form the upper chamber, and with the increase of TMP concentration this migration effect were enhanced.Conclusion:1.This experiment successfully replicated the MCAO cerebral ischemia model.AC can improve the recovery of neurological function follwing cerebral isehemia in rats, also can significantly reduce cerebral ischemia areas.2.AC can significantly promote NSCs proliferation, migration, differentiation, but R and NR on NSCs proliferation, migration, differentiation capacity diminished. Indicate R to play a role in the entire compound.Its mechanism is to promote the SDF-1, of CXCR4and VEGF in, BDNF and Reelin protein upregulation. Thus on this basis that promote NSCs proliferation, migration, differentiation is one of guiding drugs to move upwards mechanisms.3. the migration of the two main cell was significantly enhanced in the co-cultivated system. At the same time TMP can promote chemotactic migration capacity of the two cells dose-dependently.In summary, AC has a better treatment on NSCs proliferation, migration and differentiation of MCAO rats, and play an important role as Assistant and Guiding, especially take significant role in NSCs migrating to the ischemic area around, those maybe the biological basis of R act as guiding herb.