壮通饮对缺氧/复氧诱导的HUVEC/U251细胞屏障损伤的影响

Effect of Zhuangtongyin on permeability of blood-brain barrier and expression of ET-1 after ischemic brain injury

目的:观察壮通饮(ZTY)对缺氧/复氧诱导的人脐静脉血管内皮细胞(HUVEC)和人星形细胞瘤U251细胞损伤模型的作用,探讨壮药保护血脑屏障的相关机制。方法:将8只SD大鼠随机分为4组:空白组(蒸馏水)及低、中、高剂量(3.74、7.48和14.96 g/kg)ZTY含药血清组,每天1次,连续7 d灌胃饲养,获取含药血清。将HUVEC和U251细胞共培养,构建缺氧/复氧诱导的体外细胞屏障损伤模型。将细胞分为5组:空白组、模型组及低、中、高剂量ZTY含药血清组。RT-qPCR定量检测各组HUVEC中基质金属蛋白酶2(MMP2)、MMP3、MMP9和内皮素1(ET-1)的mRNA表达水平;Western blot检测各组HUVEC中紧密连接蛋白(TJPs)和ET-1蛋白的表达水平。结果:与模型组比较,给药组的跨内皮细胞电阻显著增大(P<0.05),MMP2、MMP3、MMP9和ET-1的mRNA表达水平显著降低(P<0.05),TJPs的表达水平显著升高(P<0.05),ET-1蛋白表达水平显著降低(P<0.05)。结论:ZTY通过上调TJPs、下调MMPs和ET-1表达而减轻缺氧/复氧对HUVEC/U251细胞屏障的损伤。

AIM:To investigate the effect of Zhuangtongyin(ZTY)on the permeability of blood-brain barrier(BBB)after cerebral ischemia injury.METHODS:Eight 8-week-old male SD rats were randomly divided into 4 groups:blank group(distilled water),and low-,middle-and high-dose(3. 74,7. 48 and 14. 96 g/kg)ZTY groups,once a day for7 consecutive days. On the 8 th day,blood was taken from abdominal aorta to acquire the drug-containing serum. Human umbilical vein endothelial cells(HUVEC)and human astrocytoma U251 cells were co-cultured to establish hypoxia/reoxygenation BBB model in vitro. The cells were randomly divided into 5 groups:blank group,model group,and low-,middle-and high-dose ZTY-containing serum treatment groups. After 24 h of treatment,the mRNA levels of matrix metalloproteinase 2(MMP2),MMP3,MMP9 and endothelin-1(ET-1)in the HUVEC were detected by RT-qPCR. The protein levels of tight junction proteins(TJPs)and ET-1 in the HUVEC were detected by Western blot.RESULTS:Compared with model group,the transendothelial electric resistance in ZTY-containing serum treatment groups was increased significantly. The mRNA expression levels of MMP2,MMP3,MMP9 and ET-1,and the protein level of ET-1 were decreased,while the protein levels of TJPs were increased after treatment with ZTY-containing serum.CONCLUSION:Zhuangtongyin reduced the damage of BBB after ischemic brain injury through up-regulating TJPs and decreased MMPs and ET-1.