| Objective:To investigate the effect and mechanism of butylphthalide on angiogenesis and neurological function improvement in rats of permanent middle cerebral artery occlusion over 48 hours after ischemia.Methods:Seventy two adult male Sprague-Dawley rats weighing 280 to 330 g were randomly divided into four groups:(1)sham operation group(Sham group,n=18),(2)permanent middle cerebral artery occlusion model(p MCAO group,n=18),(3)butylphthalide drug intervention group(NBP group,n=18)and(4)butylphthalide + PI3K-AKT pathway inhibitor LY294002 group(NBP+LY group,n=18).The sham group and the p MCAO group were intraperitoneally injected with equal volume of normal saline,and the other two groups were intraperitoneally injected with butylphthalide(6mg/kg/day).The four groups began to use the drug 48 hours after modeling,and was continuously administered for 7 days.The PI3K-AKT pathway inhibitor LY294002(5mg/kg)was given intraperitoneally in the inhibitor group 30 minutes before modeling.Each group received Longa score 1 day after modeling,and those with the score of 1-3 were included in the group.After 7 days of administration,the modified neurological deficit score(m NSS score)was performed,and the brain was decapitated.The area of cerebral infarction was measured by TTC staining.Western blot(WB)and immunohistochemical(IHC)were used to detect angiogenesis related proteins(VEGF,α-SMA and CD31),apoptosis related proteins(Bax and Bcl-2)and PI3K-AKT signaling pathway related proteins(p-AKT).Results:1.Modified neurological severity score(m NSS score): after 7 days of administration,the m NSS score of NBP group was lower than that of p MCAO group(NBP 8.33±0.58 VS p MCAO 12.67±0.58,P<0.05),and the m NSS score of NBP group was lower than NBP+LY group(NBP 8.33±0.58 VS NBP+LY 10.67±0.58,P<0.05).2.Infarction area: the proportion of total infarct area in the NBP group was smaller than that in the p MCAO group(NBP 14.79±0.13 VS p MCAO 36.42±1.50,P<0.05),and the proportion of total infarct area in the NBP group was smaller than that in the NBP+LY group(NBP 14.79±0.13 VS NBP+LY 22.52±0.30,P<0.05).3.Expression level of angiogenesis related proteinsThe results of immunohistochemistry and western blot analysis showed that compared with the sham group,the expression level of VEGF,α-SMA and CD31 in the penumbra and peripheral area of brain tissue in p MCAO group,NBP group and NBP+LY group were significantly increased(P<0.05).And the expression level of NBP group was higher than p MCAO group and NBP+LY group(P<0.05).4.Expression level of apoptosis related proteinsThe results of immunohistochemistry and western blot analysis showed that compared with the sham group,the expression level of pro-apoptotic protein Bax in the ischemic penumbra and peripheral areas of brain tissue in the p MCAO group,NBP group and NBP+LY group were significantly increased(P<0.05).The expression level of Bax in NBP group was lower than that in p MCAO group and NBP+LY group(P<0.05).However,the expression level of anti-apoptotic protein Bcl-2 in NBP group was higher than that in p MCAO group and NBP + LY group(P<0.05).5.Expression level of PI3K-AKT signaling pathway proteinsThe results of immunohistochemistry and western blot analysis showed that compared with the sham group,the expression level of p-AKT in the penumbra and peripheral area of infarct ischemia in p MCAO group,NBP group and NBP+LY group were significantly increased(P<0.05).And the expression of NBP group was higher than p MCAO group and NBP+LY group(P<0.05).Conclusion:In rats with permanent middle cerebral artery occlusion treated with butylphthalide after 48 hours of ischemia,it can reduce neurological deficit symptoms,reduce the area of cerebral infarction,promote angiogenesis in the ischemic penumbra and peripheral areas,and reduce cell apoptosis.The mechanism is related to the promotion of angiogenesis and inhibition of apoptosis by PI3K-AKT signaling pathway. |
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