Protective Mechanism Of Butylphthalide On Ischemic Stroke Based On Autophagy And PI3k-Akt-mTOR Signaling Pathway

Objective: To investigate the effects of butylphthalide on cerebral infarction volume and the recovery of neurological function in photothrombotic ischemia mice.Methods:C57BL/6J mice were divided into three groups: sham group,vehicle group and NBP group.Before the photothrombotic ischemia mouse model was established,the mice were trained to rotate for three days.After photothrombotic ischemia,vehicle group and NBP group were given normal saline and 2 mg/ml NBP solution,respectively,and 10 ul/g via intraperitoneal injection.Before and 1,3,7,14,21 and28 days after the infarction,the recovery of neural function in mice was assessed by m NSS scale and rotarod test.On the 7th day,the volume of cerebral infarction in mice was measured by Nissl staining.Results:Compared with vehicle group,the m NSS score of NBP group was lower on the 14 th,21st and 28 th day after stroke(P < 0.05);the latency of rotarod test in vehicle group was lower than that in NBP group on the 21 st and 28 th day after stroke(P < 0.05);the average ration of the infarct volume of NBP group was smaller than that in vehicle group on the 7th day(P < 0.05).Conclusion:NBP administration can significantly improve the neurological function after two weeks of photothrombotic ischemia and reduce the infarct volume one week after infarction.Objective: To verify whether NBP administration after photothrombotic ischemia can promote neurogenesis and angiogenesis in the SVZ ischemic penumbra;and monitor the autophagic activity in cerebral infarction mice after NBP intervention.Methods:C57BL/6J mice were divided into four groups: sham group,vehicle group,NBP group and NBP+3-MA group.The vehicle group was given saline on the first day after stroke,and the NBP and NBP+3-MA groups were given NBP solution.In addition,3-MA,an autophagy inhibitor,was also given in NPB+3-MA group.Brdu was administered for one week after the infarction.Immunofluorescence staining was performed 7 days later to observe the number of Brdu,DCX,CD31 and Neu N positive cells.Brdu+/DCX+ cells in the SVZ represent immature neurons;Brdu+/CD31+ cells in the ischemic penumbra represent neovascular endothelial cells,and Neu N+ cells can evaluate survival neurons in the ischemic penumbra.Fourteen days after infarction,the expression of autophagy related proteins P62,Beclin-1 and LC3 B was detected by Western blot.Results:DCX+/Brdu+cells in SVZ region of the NBP and NBP+3-MA group mice were much more than those in vehicle group(P<0.05),and DCX+/Brdu+cells in NBP group were more than those in NBP+3-MA group(P<0.05).Compared with vehicle group and NBP+3-MA group,the number of Brdu+/CD31+ and Neu N+/DAPI+ cells in the ischemic penumbra was significantly higher in NBP group(P < 0.05),but there was no significant difference between vehicle group and NBP+3-MA group(P > 0.05).WB showed that compared with vehicle group,the expression of Beclin-1 and LC3II/I was significantly higher and P62 was significantly lower in NBP group(P < 0.05);compared with NBP group,the expression of LC3 II/I in NBP+3-MA group was significantly lower and the expression of P62 was higher(P < 0.05).Conclusion:NBP may improve the survival of neurons in the ischemic penumbra and promote angiogenesis and neurogenesis by promoting autophagy in mice after photothrombotic ischemia.This protective effect can be partially inhibited by the autophagy inhibitor 3-MA.Objective: To investigate the role of the classical PI3K-Akt-m TOR signaling pathway in the protective effect of NBP on brain microvascular endothelial cells(BMECs)after glucose oxygen deprivation and reperfusion injury.Methods : BMECs were divided into four groups: control group,glucose oxygen deprivation and reperfusion group(OGD),glucose oxygen deprivation and reperfusion + NBP group(NBP),glucose oxygen deprivation and reperfusion + NBP+ LY294002 group(OGD/NL).Different groups of BMEC were given different interventions before OGD.NBP and OGD/NL groups were pretreated with NBP and NBP+LY294002 for 12 hours before OGD.MTT assay was used to observe the cell viability after OGD.Western blot was used to detect the expression of autophagy related proteins and PI3K-Akt-m TOR signaling pathway.Results:Compared with OGD group,NBP pretreatment at different concentrations(1u M,10 u M and 100 u M)could improve the cell viability after OGD(P < 0.05),but the survival rate of cells in the OGD/NL group was significantly lower than that of NBP and OGD groups(P < 0.05).Compared with control group,cells in OGD group and OGD/NL group showed higher expression of Beclin-1 and LC3 II/I(P < 0.05).The expressions of Beclin-1 and LC3 II/I in NBP and OGD/NL groups were significantly lower than those in OGD group(P < 0.05),while the expression of P62 protein was significantly higher(P < 0.05).The expression of P62 in OGD/NL group was lower than that in NBP group(P < 0.05).The ratios of p-m TOR/m TOR and p-Akt/Akt in NBP group were higher than those in OGD group(P < 0.05).Compared with OGD/NL group,the expression of PI3 K in NBP group was higher than that in OGD/NL group(P < 0.05).Conclusion : Glucose oxygen deprivation and reperfusion injury can increase intracellular autophagy activity,and NBP pretreatment can protect cells and reduce autophagy activity by activating PI3K-Akt-m TOR pathway.