摘要
目的观察寒痉汤对冷刺激诱导血管平滑肌细胞氧化应激的保护作用及其作用机制。方法将实验细胞分为空白组、模型组、寒痉汤组及富马酸二甲酯组,以富马酸二甲酯为阳性对照,各组血管平滑肌细胞(A7r5)培养24 h后,4℃冷刺激4 h。采用CCK-8法检测各组细胞活力;采用化学法检测细胞超氧化物歧化酶(SOD)、丙二醛(MDA)表达水平;采用DCFH-DA荧光法检测细胞活性氧(ROS)的表达;采用Elisa法检测细胞白细胞介素1β(IL-1β)、核苷酸结合寡聚结构域样受体蛋白3(NLRP3)表达水平;采用免疫印迹法检测细胞核因子E2相关因子2(Nrf2)、NAD(P)H醌氧化还原酶1(NQO1)、核因子κB(NF-κB)、人核因子κB抑制蛋白α(IκBα)蛋白表达水平;采用实时荧光定量PCR技术检测NQO1 mRNA、NF-κB mRNA表达水平。结果与空白组相比,模型组细胞活力、SOD表达降低(P<0.01),ROS、MDA、IL-1β、NLRP3表达升高(P<0.01);NF-κB、IκBα蛋白表达升高(P<0.01),Nrf2、NQO1蛋白表达降低(P<0.01);NF-κB mRNA表达升高(P<0.01),NQO1 mRNA表达降低(P<0.01)。与模型组相比,寒痉汤组细胞活力升高(P<0.01),SOD表达升高(P<0.01);ROS表达降低(P<0.01);MDA表达降低(P<0.05),IL-1β、NLRP3表达降低(P<0.01),Nrf2、NQO1蛋白表达升高(P<0.01),NF-κB、IκBα蛋白表达降低(P<0.05,P<0.01);NF-κB mRNA表达降低(P<0.01),NQO1 mRNA表达升高(P<0.05)。结论寒痉汤提高冷刺激主动脉平滑肌细胞活力,调节细胞ROS表达,抑制炎症因子表达,改善氧化应激反应,其机制可能与Nrf2/NF-κB通路有关。
Objective To observe the effects and mechanism of Hanjingtang on the oxidative stress induced by cold stimulation in aortic vascular smooth muscle cells.Methods The experimental cells were divided into blank control group,model group,Hanjingtang(HJT)group and dimethyl fumarate(DMF)group.The DMF group was defined as the positive control.After vascular smooth muscle cells(A7r5)were cultured for 24 hours,they received cold stimulation at 4℃for 4 hours.Cell viability was detected with CCK-8;expressions of superoxide dismutase(SOD)and malonaldehyde(MDA)were determined with immunochemistry;expression of active oxygen(ROS)was detected with DCFH-DA fluorescence;expressions of interleukin-1β(IL-1β)and nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)were assessed with Elisa;protein expressions of cellular nuclear factor E2-associated factor 2(Nrf 2),NAD(P)H quinone oxidoreductase 1(NQO1),nuclear factorκB(NF-κB),and human nuclear factorκB inhibitor proteinα(IκBα)were determined with Western blotting;mRNA expressions of NQO1 and NF-κB were detected with quantitative real-time PCR.Results Compared with the blank control group,the model group had decreased cell viability and SOD(P<0.01);increased ROS,MDA,IL-1βand NLRP3 levels;increased protein expressions of NF-κB and IκBα,but decreased protein expressions of Nrf2 and NQO1(P<0.01);increased mRNA expression of NF-κB,but decreased mRNA expression of NQO1(P<0.01).Compared with the model group,the HJT group had increased cell viability and SOD(P<0.01);decreased ROS,IL-1β,NLRP3(P<0.01),and MDA(P<0.05)levels;increased protein expressions of Nrf2 and NQO1(P<0.01),but decreased protein expressions of NF-κB and IκBα(P<0.05,P<0.01);decreased mRNA expression of NF-κB(P<0.01),but increased mRNA expression of NQO1(P<0.05).Conclusion The mechanism of enhancing cold-stimulated aortic smooth muscle cell vitality,regulating cellular ROS expression,inhibiting inflammatory factor expression,and improving oxidative stress response may be related to Nrf2/NF-κB pathway.
作者
刘洋
陈贵海
LIU Yang;CHEN Guihai(College of Traditional Chinese Medicine,Shandong University of Traditional Chinese Medicine,Jinan 250355,Shandong,China;Basic Medical School,Guangxi University of Traditional Chinese Medicine,Nanning 530000,Guangxi,China)
出处
《山东大学学报（医学版）》
CAS
北大核心
2023年第8期24-30,共7页
Journal of Shandong University:Health Sciences
基金
2019年广西中医药大学“国家自然科学基金”结余经费资助课题(81460690)。
关键词
冷刺激
氧化应激
寒痉汤
核因子E2相关因子2
核因子ΚB
Cold stimulation
Oxidative stress
Hanjingtang
Nuclear factor E2-associated factor 2
Nuclear factorκB
