清热化瘀方对脑缺血再灌注大鼠脑组织氧化应激反应的影响及机制

The effect and mechanism of Qingre Huayu Decoction on oxidative stress in rats with cerebral ischemia-reperfusion injury

目的探讨清热化瘀方对脑缺血再灌注(I/R)大鼠脑组织氧化应激反应的影响及其作用机制。方法32只SD大鼠分为正常组、假手术组、模型组及清热化瘀方组4组,每组8只。除正常组外,其余各组均采用线栓法进行处理;大鼠造模后正常饲养,自由饮水;清热化瘀方组给予清热化瘀方(1.4 mL/100 g,bid)灌胃,其余各组均给予等体积生理盐水灌胃,4天后处死。采用RT-qPCR、Western blot和ELISA法分别检测缺血灶脑组织中miR-18a-3p表达、Keap1、Nox-2、Nox-4、HO-1、SOD2 mRNA和蛋白表达、Nrf2核转位及ROS水平。双荧光素酶报告基因检测miR-18a-3p与Keap1的相互作用。结果与正常组相比,模型组缺血灶脑组织中miR-18a-3p、HO-1、SOD2表达及Nrf2核转位显著降低,Nox-2、Nox-4、Keap1表达及ROS水平显著升高(均P<0.05);与模型组相比,清热化瘀方组缺血灶脑组织中miR-18a-3p、HO-1、SOD2表达及Nrf2核转位显著升高,Nox-2、Nox-4、Keap1表达和ROS水平显著降低(均P<0.05);假手术组与正常组的检测结果比较,差异无统计学意义(P>0.05)。双荧光素酶报告基因检测结果证实,miR-18a-3p靶向Keap1基因的3′UTR。结论清热化瘀方可抑制脑缺血再灌注大鼠脑组织氧化应激反应,其机制可能与促进缺血灶脑组织miR-18a-3p表达,进而抑制Keap1表达,促进Nrf2核转位有关。

Objective To investigate the effect of Qingre Huayu prescription on oxidative stress in rats with cerebral ischemia-reperfusion injury(I/R)and further explore its mechanism.Methods SD rats were randomly divided into normal group,sham operation group,model group and Qingre Huayu prescription group,with 8 rats in each group.Except for the normal group,all the other groups were treated by suture method.The rats were fed normally and drank freely after modeling.The Qingre Huayu prescription group was given the Qingre Huayu prescription(1.4 mL/100g,bid,i.g.),and the other groups were given the same volume of normal saline by gavage,and were killed after 4 days.The expression of miR-18a-3p,Keap1,Nox-2,Nox-4,HO-1,SOD2 mRNA and protein,Nrf2 nuclear translocation and ROS levels in ischemic brain tissue were observed by RT-qPCR,Western blot and ELISA separately.The interaction between miR-18a-3p and Keap1 was detected by the double luciferase reporter gene.Results As compared with the normal group,the expressions of miR-18a-3p,HO-1,SOD2 and Nrf2 nuclear translocation in ischemic brain tissues of the model group were significantly decreased(P<0.05),while the expressions of Nox-2,Nox-4,Keap1 and ROS levels were significantly increased(P<0.05).As compared with the model group,the expressions of miR-18a-3p,HO-1,SOD2 and Nrf2 nuclear translocation in the ischemic brain tissues of the Qingre Huayu prescription group were significantly increased(P<0.05),and the expressions of Nox-2,Nox-4,Keap1 and ROS levels were significantly decreased(P<0.05).There was no significant difference in the results between the sham operation group and the normal group(P>0.05).The results of dual luciferase reporter gene testing confirmed that miR-18a-3p targets the 3′UTR of Keap1 gene.Conclusion Qingre Huayu prescription can inhibit the oxidative stress response in brain tissues of cerebral I/R rats,and the mechanism may be related to promoting the expression of miR-18a-3p in ischemic brain tissues,thereby inhibiting the expression of Keap1 and promoting the nuclear translocation of Nrf2.