摘要
目的:探讨松花粉对过氧化氢(H_2O_2)诱导人肝癌HepG2细胞应激性氧化损伤的保护作用。方法:将HepG2细胞分为正常组,H_2O_2模型组和样品干预组。样品干预组用不同浓度的松花粉预处理HepG2细胞12 h,H_2O_2模型组和样品干预组用400μmol·L^(-1)过氧化氢氧化损伤细胞2 h,产生氧化应激损伤。采用噻唑蓝(MTT)比色法检测HepG2细胞活力;微板法检测细胞内活性氧(ROS),超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px)活性,乳酸脱氢酶(LDH)及丙二醛(MDA)含量;蛋白免疫印迹法(Western blot)检测抗氧化通路中关键基因核因子E2相关因子2(Nrf2),Kelch样环氧氯丙烷相关蛋白1(Keap1),谷氨酸半胱氨酸连接酶(GCL)和血红素氧合酶-1(HO-1)蛋白的表达水平,以评价松花粉对HepG2细胞氧化应激损伤的保护作用。结果:MTT结果显示,与正常组比较,松花粉(80,40,20,10,5 mg·L^(-1))对HepG2细胞没有毒性;H_2O_2模型组中ROS,LDH及MDA的水平显著升高(P<0.01),SOD和GSH-Px的活性显著降低(P<0.01)。与H_2O_2模型组比较,松花粉干预组中ROS,LDH及MDA的水平显著降低(P<0.05,P<0.01),SOD和GSH-Px的活性显著升高(P<0.05,P<0.01)。Western blot结果显示,松花粉显著上调Nrf2,HO-1和GCL的蛋白表达,并下调Keap1的蛋白表达。结论:质量浓度5~20 mg·L^(-1)松花粉可有效保护400μmol·L^(-1)H_2O_2对HepG2细胞应激性氧化损伤。其机制可能与调节SOD,GSH-Px活性,ROS,LDH,MDA水平有关,同时与调控抗氧化通路中关键基因Nrf2,Keap 1,HO-1,GCL蛋白的表达水平有关。
Objective： To investigate the protective effect of Pollen Pini on oxidative damage of HepG2 cells induced by H2O2. Method： HepG2 cells were divided into normal control group, H2O2 model group and intervention group. HepG2 cells of the intervention group were pretreated with different concentrations of Pollen Pini for 12 h. Then, to produce oxidative stress, the H2O2 model group and the intervention group were treated with 400 μmol·L^-1 H2O2 to damage cells for 2 h. Cell viability was measured by methylthiazolyl tetrazolium （MTT） assay. To evaluate the protective effect of Pini Pollen on HepG2 cells, cell viability, intracellular reactive oxygen species （ROS）, superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） activities, lactate dehydrogenase （LDH） and malondialdehyde （MDA） contents were detected. In addition, the expressions of key genes nuclear factor-E2-related factor 2 （Nrf2）, Kelch-like epichlorohydrin-associated protein 1 （Keap 1）, heme oxygenase-1 （HO-1）, glutamate-cysteine ligase （GCL） in antioxidant pathway were determined by western blot. Result： MTT results showed that, compared with normal control group, the cytotoxicity of Pollen Pini groups （80, 40, 20, 10, 5 mg·L^-1） were non-toxic. The contents of ROS, LDH and MDA in the H2O2 model group were significantly increased （P〈0.01）, while the activities of SOD and GSH-Px were significantly depressed （P〈0.01）. Compared with the H2O2 model group, the contents of ROS, LDH and MDA in the intervention group were significantly reduced （P〈0.05, P〈0.01）, whereas the activities of SOD and GSH-Px were significantly increased （P〈0.05, P〈0.01）. Western blot results indicated that pollen pini up-regulated protein expressions of Nrf2, HO-1 and GCL, but down-regulated the expression of Keap1 protein. Conclusion： The 5-20 mg·L^-1 of Pollen Pini can effectively protect 400 μmol·L^-1 H2O2-induced oxidative stress damage in HepG2 cells. The mechanism may be related to regulation of the activity of SOD, GSH-Px activity, ROS, LDH and MDA levels, and protein expressions of key genes Nrf2, Keap 1, HO-1, and GCL in the antioxidant pathway.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2017年第19期167-173,共7页
Chinese Journal of Experimental Traditional Medical Formulae
