Based On The ERK1/2 Signal Pathway To Explore The Effect Of Shennao Fuyuan Decoction On The Proliferation And Differentiation Of NSCs After Cerebral Ischemic Injury And Its Mechanism

Objective:1.Cell experiment: To investigate the effect and mechanism of Shennao Fuyuan Decoction on the proliferation and differentiation of embryonic rat NSCs after OGD intervention.2.Animal experiment:To investigate the effect and mechanism of Shennao Fuyuan Decoction on the proliferation and neuronal differentiation of endogenous NSCs after ischemia injury.Methods:1.Cell experiment: NSCs was cultured from the cerebral cortex of fetal rats for 14 days.OGD intervention was used to simulates the conditions of cerebral ischemia.10 uM BrdU was added into the proliferation medium for the intervention of NSCs for 24 h in the proliferation experiment.Differentiation medium was used to interpose NSCs for 7 days in the differentiation experiment.NSCs were divided into four groups: normal group,model group,Chinese medicine(CM)group and inhibitor group.CCK-8 was used to detect cell proliferation activity.The expression of BrdU,MAP-2,GFAP was detected with immunofluorescence.Western blot was used to detect the protein expression of MAP-2?GFAP?p-ERK1/2.2.Animal experiment: adult SD rats were randomly divided into normal group,model group,CM group and inhibitor group.MCAO operation was used to simulate of the conditions of ischemic stroke.Neurological deficit scores and infarct volume were used to evaluate tissue repair.The expression of Nestin and MAP-2 in hippocampe and subventricular zone was detected with immunohistochemical.Western blot was used to detect the protein expression of p-ERK1/2.Results:1.Cell experiment:(1)CCK-8 results showed that after OGD intervention of 24 h cell proliferation activity in CM group were significantly higher than those in the model group and inhibitor group,there was statistical difference among the groups(P<0.05 ? P<0.01).(2)In the proliferation experiment,immunofluorescence positive cell percentage of BrdU in CM group was obviously higher than that in model group and inhibitor group after Shennao Fuyuan Decoction's intervention for 24 h.There was statistical difference among the groups(P<0.05 ?P<0.01).(3)Western bolt detection in proliferation experiment: the protein expression of p-ERK1/2 in CM group was significantly higher than the model group and inhibitor group.There was significant statistical difference among the groups(P<0.01).(4)In the differentiation experiment,immunofluorescence positive cell percentage of MAP-2 in CM group was significantly higher than the model group and inhibitor group.There was significant difference among the groups(P<0.01).Immunofluorescence positive cell percentage of GFAP in CM group was significantly lower than the model group and inhibitor group.There was significant statistical difference among the groups(P<0.01).(5)Western bolt detection in differentiation experiment: the protein expression of MAP-2 and p-ERK1/2 in CM group was significantly higher than the model group and inhibitor group,and the protein expression of GFAP in CM group was significantly lower than the model group and inhibitor group.There was significant statistical difference among the groups(P<0.01).2.Animal experiment:(1)Neurological deficit score: 3,7,14 days after MCAO operation,the neurological deficit score of CM group was significantly lower than that of the model group and inhibitor group at the same time point.There was statistical difference among the groups(P<0.05 ? P<0.01).(2)The volume of cerebral infarction: 3,7,14 days after treatment,the volume of cerebral infarction in CM group was less than that in the model group and inhibitor group at the same time point.There was statistical difference among the groups(P<0.05 ? P<0.01).(3)Endogenous NSCs proliferation: the number of Nestin positive cells in CM group at each time point was significantly higher than that in the model group and inhibitor group.There was significant statistical difference among the groups(P<0.01).(4)Neuronal differentiation: the MAP-2 positive cells in CM group at each time point were significantly higher than those in the model group and inhibitor group.There was significant statistical difference among the groups(P<0.01).(5)The expression of pERK1/2 protein: the expression of p-ERK1/2 protein in CM group was higher than that in the model group and inhibitor group at 3,7 and 14 days after operation.There was significant statistical difference between the two groups(P<0.01).Conclusion:1.Shennao Fuyuan Decoction promotes the proliferation of NSCs and leads NSCs differentiate into neurons in vitro,inhibits of astrocyte differentiation,by the regulation of ERK1/2 signal pathway.2.Shennao Fuyuan Decoction promotes the proliferation of endogenous NSCs,leads NSCs differentiate into neurons,protect the brain,by the regulation of ERK1/2 signal pathway.