黄芪苷保护缺血损伤BMECs的作用机制研究

Mechanism of astragaloside protecting bmecs from ischemia injury

目的探讨黄芪苷通过NO/Cav-1/MMP-9/Caspase-3信号通路保护缺血损伤脑微血管内皮细胞(BMECs)的作用机制。方法采集出生2 w清洁级SD大鼠大脑皮质,采用连续消化法获得大脑皮质细胞,取生长状态良好的第2~3代BMECs细胞进行研究。取等量的BMECs细胞,加入缺血液的为缺血组,加入缺血液+10μmol/L黄芪苷为低浓度实验组,加入缺血液+50μmol/L黄芪苷为中浓度实验组,加入缺血液+100μmol/L黄芪苷为高浓度实验组,加入等量PBS为空白对照组。MTT法检测BMECs增殖,Western-blot法检测Caspase-3和MMP-9表达水平,试剂盒检测Cav-1和NO表达水平。结果与空白对照组比较,缺血组、低浓度实验组、中浓度实验组、高浓度实验组BMECs细胞增殖水平均明显下降,差异均有统计学意义(t=5.38、4.92、4.35、3.89,均P<0.05);与缺血组比较,低浓度实验组、中浓度实验组、高浓度实验组、空白对照组BMECs细胞增殖水平均上升,除低浓度实验组比较差异无统计学意义(P>0.05)外,中浓度实验组、高浓度实验组、空白对照组差异均有统计学意义(t=5.51、6.01、5.38,均P<0.05)。空白对照组Caspase-3和MMP-9表达水平分别为(0.14±0.15)、(0.47±0.15),缺血组Caspase-3和MMP-9表达水平分别为(0.87±0.12)、(1.36±0.10),高浓度组Caspase-3和MMP-9表达水平分别为(0.28±0.02)、(0.58±0.12),中浓度组Caspase-3和MMP-9表达水平分别为(0.33±0.06)、(0.79±0.04),低浓度组Caspase-3和MMP-9表达水平分别为(0.38±0.08)、(1.17±0.07)。与空白对照组比较,缺血组、低浓度实验组、中浓度实验组、高浓度实验组Caspase-3和MMP-9表达水平均明显升高,差异均有统计学意义(tCaspase-3=70.12、24.70、23.95、20.63,tMMP-9=25.79、22.26、18.18、3.11,均P<0.05);与缺血组比较,低浓度实验组、中浓度实验组、高浓度实验组、空白对照组Caspase-3和MMP-9表达水平明显下降,差异均有统计学意义(tCaspase-3=43.81、52.95、66.65、70.12,tMMP-9=4.09、17.81、28.08、25.79,均P<0.05)。与空白对照组比较,缺血组、低浓度实验组、中浓度实验组、高浓度实验组Cav-1表达水平均明显下降、NO表达水平均明显升高,差异均有统计学意义(tCav-1=17.37、8.68、11.58、8.68,tNO=-10.10、-9.13、-4.13、-2.88,均P<0.05);与缺血组比较,低浓度实验组、中浓度实验组、高浓度实验组、空白对照组Cav-1表达水平均明显升高、NO表达水平均明显下降,差异均有统计学意义(tCav-1=-6.74、-11.02、-16.53、-17.37,tNO=4.83、7.63、9.01、10.10,均P<0.05)。结论黄芪苷可能通过调节Caspase-3、MMP-9、Cav-1及NO表达水平来发挥对BMECs的保护作用。

Objective To investigate the mechanism by which astragaloside protects brain microvascular endothelial cells(bmecs)from ischemic injury through the NO/Cav-1/MMP-9/caspase-3 signaling pathway.Methods Cortical brains were collected from newborn 2-W clean grade SD rats,and serial digestion was used to obtain cortical cells,from passage 2 to 3 bmecs that were in good growth condition for study.Equal amounts of BMECs cells were collected,addingischemic solutionas the ischemic group,adding ischemic solution and+10μmol/L astragalo side as the low concentration experi-mental group,adding ischemic solution and+50μmol/L astragalo side as the medium concentration experimental group,adding ischemic solution and+100μmol/L astragalo side as the high concentration experimental group,and adding equal amounts of PBS as the blank control group.The proliferation of bmecs was detected by MTT assay,the expression levels of Caspase-3 and MMP-9 were detected by Western blot assay,and the expression levels of Cav-1 and no were detected by kits.Results Compared with the blank control group,the levels of bmecs cell proliferation in the ischemic group,the low concentration experimental group,the middle concentration experimental group,and the high concentration experimental group all decreased significantly,all differences were statistically significant(t=5.38,4.92,4.35,3.89,all P<0.05);Compared with the ischemic group,the levels of bmecs cell proliferation in the low concentration experimental group,the middle concentration experimental group,the high concentration experimental group,and the blank control group all rose,except for the low concentration experimental group,which had no statistical significance(P>0.05),the differences in the medium concentration experimental group,high concentration experimental group,and blank control group were all statisti-cally significant(t=5.51,6.01,5.38,all P<0.05).The expression levels of Caspase-3 and MMP-9 were(0.14±0.15),(0.47±0.15)in the blank control group;(0.87±0.12),(1.36±0.10)in the ischemia group;(0.28±0.02),(0.58±0.12)in the high concentration group;(0.33±0.06),(0.79±0.04)in the middle concentration group;(0.38±0.08),(1.17±0.07)in the low concentration group.Compared with the blank control group,the expression levels of Caspase-3 and MMP-9 in the ischemia group,the low concentration experimental group,the middle concentration experimental group,and the high concentration experimental group were all significantly increased,all differences were statistically sig-nificant(tCaspase-3=70.12,24.70,23.95,20.63,tMMP-9=25.79,22.26,18.18,3.11,all P<0.05);Compared with the ischemic group,the expression levels of Caspase-3 and MMP-9 in the experimental group with low concentration,the experimental group with medium concentration,the experimental group with high concentration,and the blank control group decreased significantly,all differences were statistically significant(tCaspase-3=43.81,52.95,66.65,70.12,tMMP-9=4.09,17.81,28.08,25.79,all P<0.05).Compared with the blank control group,the expression levels of Cav-1 in the ischemic group,the low concentration experimental group,the middle concentration experimental group,and the high concentration experimental group all decreased significantly,and the expression levels of no increased significantly,all with statistical significance(tCav-1=17.37,8.68,11.58,8.68,tNO=-10.10,-9.13,-4.13,-2.88,all P<0.05);When compared with the ischemic group,the expression levels of Cav-1 in the low-,medium-,high-,and blank control groups were all significantly in-creased,and the expression levels of no were all significantly decreased,all with statistical significance(tCav-1=-6.74,-11.02,-16.53,-17.37,tNO=4.83,7.63,9.01,10.10,all P<0.05).Conclusion Astragaloside may exert protective effects on bmecs by regulating Caspase-3,MMP-9,Cav-1 and no expression levels.