β-细辛醚对氧糖剥夺/复糖复氧诱导星形胶质细胞损伤的保护作用及机制研究

Protection effect and mechanism of β-asarone on astrocyte injury induced by oxygen/glucose deprivation and reintroduction

目的 探讨石菖蒲有效成分β-细辛醚对氧糖剥夺/复糖复氧损伤星形胶质细胞的保护作用及机制。方法 将星形胶质细胞分为5组:对照组、模型组、β-细辛醚(50μg/mL)组、核因子-E2相关因子2(Nrf2)抑制剂(ML385,5μmol/L)组、β-细辛醚(50μg/mL)+ML385(5μmol/L)组。CCK-8检测细胞存活率,流式细胞术测定细胞凋亡水平,以DCFH-DA法检测细胞内活性氧(ROS)水平,试剂盒检测细胞氧化应激因子[谷胱甘肽过氧化物酶(GSH-Px)、乳酸脱氢酶(LDH)、丙二醛(MDA)及超氧化物歧化酶(SOD)]及炎症因子[核因子-κB亚基p65(NF-κB p65)、白细胞介素(IL)-1β和IL-18]的水平,实时荧光定量PCR(RT-qPCR)检测Nrf2、血红素氧化酶-1(HO-1) mRNA的表达水平,Western blot检测Nrf2、醌氧化还原酶-1(NQO-1)、HO-1蛋白表达。结果 与对照组比较,模型组、ML385组及β-细辛醚+ML385组细胞凋亡率、LDH、ROS、MDA、NF-κB p56、IL-1β和IL-18水平明显升高,细胞存活率、Nrf2、NQO-1、HO-1、SOD、GSH-Px明显下降(P<0.05)。与模型组比较,β-细辛醚组及β-细辛醚+ML385组细胞凋亡率、LDH、ROS、MDA、NF-κB p56、IL-1β和IL-18明显下降,Nrf2、NQO-1、HO-1、SOD、GSH-Px明显升高,ML385组上述指标则相反(P<0.05)。与ML385组比较,β-细辛醚组及β-细辛醚+ML385组细胞存活率、Nrf2、NQO-1、HO-1、SOD、GSH-Px明显下降,Nrf2、NQO-1、HO-1、SOD、GSH-Px明显升高(P<0.05)。结论 β-细辛醚可能通过激活Nrf2/HO-1通路减轻氧糖剥夺/复糖复氧诱导的星形胶质细胞氧化应激及炎症损伤以起到保护作用。

Objective To investigate the protective effect and mechanism of β-asarone, the effective component of acorus tatarinowii, on the astrocytes damaged by oxygen and glucose deprivation and reoxygenation.Methods The astrocytes were divided into the five groups: the control group, model group, β-asarone(50 μg/mL) group, nuclear factor-E2-related factor 2(Nrf2) inhibitor(ML385,5 μmol/L) group, and β-asarone(50 μg/mL) +ML385(5 μmol/L) group.The cell survival rate was detected by CCK-8,the cell apoptosis level was determined by flow cytometry, and the intracellular reactive oxygen species(ROS) level was detected by DCFH-DA method.The levels of oxidative stress factors [glutathione peroxidase(GSH-PX),lactate dehydrogenase(LDH),malondialdehyde(MDA) and superoxide dismutase(SOD)] and inflammatory factors [nuclear factor κB subunit p65(NF-κB p65),interleukin(IL)-1β and IL-18] were detected by the kit, the real-time quantitative PCR(RT-qPCR) was used to detect the mRNA expression levels of Nrf2 and heme oxidase-1(HO-1),and Western blot was used to detect the protein expressions of Nrf2,quinone oxidoreductase-1(NQO-1) and HO-1.Results Compared with the control group, the apoptosis rate, LDH,ROS,MDA,NF-κB P56,IL-1β and IL-18 levels in the model group, ML385 group and β-asarone+ML385 group were significantly increased, while the cell survival rate, Nrf2,NQO-1,HO-1,SOD and GSH-Px were decreased significantly(P<0.05).Compared with the model group, the apoptosis rate, LDH,ROS,MDA,NF-κB p65,IL-1β and IL-18 inthe β-asarone group and β-asarone+ML385 group were significantly decreased, while Nrf2,NQO-1,HO-1,SOD and GSH-Px were significantly increased, while the ML385 group had the opposite effect(P<0.05).Compared with the ML385 group, the cell survival rate, LDH,ROS,MDA,NF-κB p56,IL-1β and IL-18 in the β-asarone group and β-asarone+ML385 group were significantly decreased, while Nrf2,NQO-1,HO-1,SOD and GSH-Px were significantly increased(P<0.05).Conclusion β-asarone may play a protective role on astrocyte injury by activating Nrf2/HO-1 signaling pathway to reduce oxidative stress and inflammatory injury induced by oxygen and glucose deprivation/reoxygenation.