Objective: To investigate the effects of blocking lactate synthesis on the HT22 cell injuries caused by hypoxia.
Methods: 2-deoxy-D-glucose (2-DG) is a non-metabolized glucose analogue that can inhibit lactate synthesis by blocking glycolysis. HT22 cells were divided into 4 groups: Control group, 2-DG group, Hypoxia group and 2-DG+Hypoxia group. The cells in control group and 2-DG treatment group were cultured in a 37℃, 5% CO2 incubator, and thecells in hypoxia group and 2-DG + Hypoxia group were cultured in a hypoxia incubator. The concentrations of 2-DG were 2.5 and 5 mmol/L, the concentration of oxygen was 0.3%, and the treatment time was 24 h. Cell activity was detected by CCK-8 assay, the levels of lactate in cell culture medium were detected by spectrophotometry, cell morphology was observed by fluorescence staining, the level of reactive oxygen species (ROS) was detected by flow cytometry, and the activities of superoxide dismutase (SOD) and catalase (CAT) were determined by enzyme activity kits. The protein expression levels of p-p38, t-p38 and β-actin were detected by Western blot.
Results: Compared with that in control group, the lactate level in culture medium and cell activity were decreased significantly (P＜0.01), the number of adherent cells was decreased, the level of ROS was increased (P＜0.01), and the enzyme activity of CAT was decreased (P＜0.05) in the 2-DG group. In the hypoxia group, the level of lactate in the culture medium was increased significantly (P＜0.01), the cell activity was decreased (P＜0.01), the number of adherent cells was decreased, the ROS levels were increased (P＜0.01), and the enzyme activities of CAT and SOD were decreased (P＜0.01 or P＜0.05). In 2-DG+Hypoxia group, the level of lactate was decreased significantly (P＜0.05), the cell viability was decreased significantly (P＜0.01), the number of cells was decreased significantly, and the ability of adhere to the wall was weakened significantly. The level of ROS was increased significantly (P＜0.01), the enzyme activities of CAT and SOD were decreased significantly (P＜0.01), the protein expression level of p-p38 was increased significantly (P＜0.05), and there was no change in t-p38. Compared with hypoxia groups, in 2-DG combined with hypoxia group, the level of lactate induced by hypoxia, the cell activity, and the enzyme activity level of CAT were decreased significantly (all P＜0.01), while the level of ROS was increased significantly (P＜ 0.01).
Conclusion: Blocking lactate can reduce the cell activity level under hypoxia and aggravate the oxidative stress injury of HT22 cells. The mechanisms may be related to increasing ROS level and activating p38 signal pathway.
目的: 探讨阻断乳酸生成对神经元HT22低氧性损伤的影响。方法: 2-脱氧-D-葡萄糖(2-DG)是一种不可代谢的葡萄糖类似物,可通过阻断糖酵解过程而抑制乳酸生成。将HT22细胞分为4组:对照组、2-DG组、Hypoxia组和2-DG+Hypoxia组。对照组与2-DG 处理组放置于37℃、5 %CO2培养箱中常规培养,Hypoxia组与2-DG+Hypoxia组放置于低氧培养箱中培养。2-DG浓度为2.5、5 mmol/L,氧气浓度为0.3%,处理时间为24 h。CCK-8法检测细胞活性,分光光度法检测细胞培养液中乳酸含量,荧光染色观察细胞形态,流式细胞术检测活性氧(ROS),酶活性试剂盒测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性,蛋白印迹(Western blot)检测p-p38, t-p38和β-actin的蛋白表达水平的变化。结果: 与对照组相比,2-DG组培养液乳酸水平与细胞活性明显降低(P＜0.01),贴壁细胞数量减少,ROS水平升高(P＜0.01),CAT酶活性降低(P＜0.05)。Hypoxia组中培养液的乳酸水平明显增加(P＜0.01),细胞活性降低(P＜0.01),贴壁细胞数量减少,ROS水平升高(P＜0.01),CAT、SOD酶活性降低(P＜ 0.01或P＜0.05),2-DG+Hypoxia组乳酸水平显著降低(P＜0.05),细胞活性显著降低(P＜0.01),细胞数量与贴壁能力明显减弱,ROS水平显著升高(P＜0.01),CAT、SOD酶活性显著降低(P＜0.01),p-p38蛋白表达水平显著升高(P＜0.05),t-p38没有变化。与Hypoxia组比较,2-DG+Hypoxia组能够抑制低氧诱导的乳酸水平升高(P＜0.01),细胞活性显著降低(P＜0.01), ROS水平显著升高(P＜0.01),CAT的酶活性显著降低(P＜0.01)。结论: 阻断乳酸生成可降低低氧下细胞活性水平,加重HT22细胞氧化应激损伤,其机制可能通过升高ROS水平、激活p38信号通路。.
Keywords: hypoxia; lactate; neurons; oxidative stress.