血管紧张素Ⅱ调控基质金属蛋白酶9的表达在蛛网膜下腔出血发病中的机制研究

Mechanism of AngⅡ regulating MMP-9 expression in pathogenesis of SAH

目的探讨血管紧张素Ⅱ(AngⅡ)调控基质金属蛋白酶9(MMP⁃9)的表达在蛛网膜下腔出血(SAH)发病中的机制。方法人脑微血管内皮细胞(HBMEC)株分为AngⅡ组、AngⅡ+SB203580组和对照组。其中AngⅡ组以100μg/L浓度AngⅡ处理90 min、2 h、4 h、6 h、12 h后取细胞上清液进行相关指标检测;AngⅡ+SB203580组以5μΜ的SB203580处理20 min后以100μg/L浓度AngⅡ处理90 min、2 h、4 h、6 h、12 h后取细胞上清液进行相关指标检测;对照组加入RPMI⁃1640培养液培养90 min、2 h、4 h、6 h、12 h后取细胞上清液进行相关指标检测。以酶联免疫吸附法(ELISA)检测各组细胞上清液MMP⁃9水平,并以实时荧光定量PCR和蛋白质印迹法检测各组细胞p38丝裂原活化蛋白激酶(p38 MAPK)的mRNA和蛋白表达水平。结果与对照组比较,AngⅡ组细胞上清液MMP⁃9[处理12 h:(4.21±1.36)μg/L比(5.81±1.72)μg/L,P<0.01]水平升高,AngⅡ+SB203580组细胞上清液MMP⁃9[处理12 h:(4.21±1.36)μg/L比(3.64±1.45)μg/L,P<0.01]水平降低(P<0.05)。与AngⅡ组比较,AngⅡ+SB203580组细胞上清液MMP⁃9水平降低(P<0.001)。与对照组比较,AngⅡ组细胞p38 MAPK的mRNA[处理12 h:(0.422±0.057)比(0.538±0.071),P<0.05)]和蛋白表达水平[(0.452±0.105)比(0.635±0.133),P<0.001]升高,AngⅡ+SB203580组细胞p38 MAPK的mRNA[处理12 h:(0.422±0.057)比(0.375±0.066),P<0.05]和蛋白表达水平[(0.452±0.105)比(0.276±0.081),P<0.001]降低(P<0.05)。与AngⅡ组比较,AngⅡ+SB203580组细胞p38 MAPK的mRNA和蛋白表达水平降低(P<0.001)。结论AngⅡ可能通过激活p38 MAPK信号通路上调MMP⁃9表达从而促进SAH发生发展。

Objective To investigate the mechanism of angiotensinⅡ(AngⅡ)regulating the expression of matrix metalloproteinase⁃9(MMP⁃9)in the pathogenesis of subarachnoid hemorrhage(SAH).Methods Human brain microvascular endothelial cell(HBMEC)lines were divided into the AngⅡgroup,the AngⅡ+SB203580 group and the control group.The AngⅡgroup was treated with 100μg/L AngⅡfor 90 minutes,2 hours,4 hours,6 hours,12 hours,then the cells supernatant was taken for the relevant indicators detection.The AngⅡ+SB203580 group was treated with 5μΜSB203580 for 20 minutes and then treated with 100μg/L AngⅡfor 90 minutes,2 hours,4 hours,6 hours,12 hours,then the cells supernatant was taken for the relevant indicators detection.The control group was added with RPMI⁃1640 medium for 90 min,2 h,4 h,6 h and 12 h,and then cell supernatant was taken for detection of related indexes.The levels of MMP⁃9 in cell supernatant were detected by ELISA method,and the expression of p38 mitogen activated protein kinase(p38 MAPK)messenger ibonucleic acid(mRNA)and protein were detected by real⁃time quantitative polymerase chain reaction and Western Blot.Results Compared with the control group,the level of MMP⁃9[intervention 12 h:(4.21±1.36)μg/L vs.(5.81±1.72)μg/L,P<0.001]in the supernatant of AngⅡgroup increased,and MMP⁃9[intervention 12 h:(4.21±1.36)μg/L vs.(3.64±1.45)μg/L,P<0.01]in the supernatant of AngⅡ+SB203580 group decreased(P<0.001).Compared with the AngⅡgroup,the level of MMP⁃9 in supernatant of AngⅡ+SB203580 group decreased(P<0.05).Compared with the control group,the mRNA[intervention 12 h:(0.422±0.057)vs.(0.538±0.071),P<0.05]and protein expression[(0.452±0.105)vs.(0.635±0.133),P<0.001]of p38 MAPK increased in AngⅡgroup cells,and the mRNA[intervention 12 h:(0.422±0.057)vs.(0.375±0.066),P<0.05]and protein expression[(0.452±0.105)vs.(0.276±0.081),P<0.001]of p38 MAPK decreased in AngⅡ+SB203580 group cells(P<0.05).Compared with the AngⅡgroup,the mRNA and protein expression of p38 MAPK in AngⅡ+SB203580 group decreased(P<0.001).Conclusion AngⅡmay promote the development of SAH by activating the p38 MAPK signaling pathway and upregulating the MMP⁃9 expression.