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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 pine pollen on oxidative stress induced by hydrogen peroxide (H_2O_2) in human hepatocellular carcinoma HepG2 cells. Methods: HepG2 cells were divided into normal group, H 2 O 2 model group and sample intervention group. In the intervention group, HepG2 cells were pretreated with different concentrations of pine pollen for 12 h. The H 2 O 2 model group and the sample intervention group were exposed to 400 μmol·L -1 H2O2 for 2 h to induce oxidative stress injury. The viability of HepG2 cells was assayed by MTT colorimetric assay. The activity of intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) Lactate dehydrogenase (LDH) and malondialdehyde (MDA). Western blot was used to detect the expression of nuclear factor E2 (Nrf2), Kelch-like epichlorohydrin-related protein 1 (Keap1), glutamic acid cysteine ligase (GCL) and heme oxygenase-1 (HO-1) protein expression levels in order to evaluate the protective effect of pine pollen on oxidative stress injury in HepG2 cells. Results: The results of MTT assay showed that pine pollen (80, 40, 20, 10, 5 mg · L -1) had no toxic effect on HepG2 cells compared with normal group. The levels of ROS, LDH and MDA in H 2 O 2 model group were significantly (P <0.01), and the activity of SOD and GSH-Px decreased significantly (P <0.01). The levels of ROS, LDH and MDA in pineal pollen intervention group were significantly lower than those in H 2 O 2 model group (P <0.05, P <0.01), and the activities of SOD and GSH-Px were significantly increased (P <0.05, P <0.01). Western blot results showed that pine pollen significantly up-regulated the protein expression of Nrf2, HO-1 and GCL, and down-regulated the protein expression of Keap1. CONCLUSION: The pine pollen of 5-20 mg · L -1 could effectively protect the HepG2 cells from oxidative stress induced by 400 μmol·L -1 H 2 O 2. The mechanism may be related to the regulation of SOD, GSH-Px activity, ROS, LDH, MDA levels, and the expression of key genes Nrf2, Keap 1, HO-1 and GCL in the antioxidant pathway.