巨自噬和分子伴侣介导自噬在内皮细胞氧化应激损伤中的特点

Characteristics of macroautophagy and molecular chaperone-mediated autophagy in oxidative stress injury of endothelial cells

目的 探究巨自噬和分子伴侣介导自噬(chaperone-mediated autophagy, CMA)这两种自噬途径在人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)氧化应激损伤中特点。方法 通过不同浓度(0、20、50、100、200μg/mL)氧化低密度脂蛋白(oxidation low lipoprotein, OX-LDL)作用HUVECs后产生的活性氧(reactive oxygen species, ROS)水平筛选诱导HUVECs氧化应激的适宜浓度。设立空白对照组(不做任何处理)、单纯抑制巨自噬组(10 mmol/L 3-MA)、氧化应激组(50μg/mL OX-LDL)和抑制巨自噬-氧化应激组(10 mmol/L 3-MA+50μg/mL OX-LDL),检测各组细胞增殖标志蛋白PCNA、凋亡标志蛋白Caspase-3、巨自噬标志蛋白Beclin-1、分子伴侣介导自噬两种标志蛋白Hsc70和Lamp-2蛋白表达,评估不同处理下HUVECs增殖、凋亡及两种自噬的变化。其中氧化应激组和抑制巨自噬-氧化应激组处理不同时间(1、3、6、12、48 h)后,采用CCK-8检测细胞增殖抑制率,Annexin-V/PI双染检测凋亡,Western blot检测两种自噬途径蛋白Beclin-1、Hsc70、Lamp-2表达,评估巨自噬抑制前后氧化应激下HUVECs的增殖、凋亡及两种自噬的时间调控特点。结果 OX-LDL诱导氧化应激下ROS浓度呈递增关系,选择50μg/mL为适宜浓度进行后续实验。与空白对照组比较,氧化应激组HUVECs Caspase-3、Beclin-1、Hsc70、Lamp-2上调,PCNA下调(P<0.05),同时随处理时间延长,48 h内细胞增殖抑制率、全期凋亡率及Lamp-2递增,Hsc70在6 h后递增,12 h内Beclin-1水平递增(P<0.05);与氧化应激组比较,抑制巨自噬-氧化应激组Caspase-3、Hsc70、Lamp-2上调,PCNA下调(P<0.05),随处理时间延长,同时段Hsc70、Lamp-2上调,且Hsc70在3 h后递增(P<0.05)。结论 巨自噬和分子伴侣介导自噬在内皮细胞氧化应激过程中先后激活,巨自噬抑制后提高分子伴侣介导自噬水平,并提前激活时间,减轻氧化应激对内皮细胞的损伤。

Objective To explore the characteristics of macroautophagy and molecular chaperone-mediated autophagy(CMA) in the oxidative stress injury of human umbilical vein endothelial cells(HUVECs). Methods After HUVECs were treated with oxidized low density lipoprotein(OX-LDL) at different concentrations(0、20、50、100、200 μg/mL), the production of reactive oxygen species(ROS) was measured to screen the optimal concentration to induce HUVECs oxidative stress. The cells were divided into 4 groups: control group(without any treatment), macroautophagy inhibition group(treated with 10 mmol/L 3-MA, specific inhibitor of macroautophagy), oxidative stress group(treated with 50 μg/mL OX-LDL), and macroautophagy inhibition-oxidative stress group(treated with 10 mmol/L 3-MA+50 μg/mL OX-LDL). The expression levels of proliferation marker protein PCNA, apoptosis marker Caspase-3, macroautophagy marker Beclin-1, and CMA markers Hsc70 and Lamp-2 were detected in each group with Western blotting. The proliferation, apoptosis, 2 kinds of autophagy under different treatments were evaluated respectively. After the cells of the oxidative stress group and macroautophagy inhibition-oxidative stress group were treated for different duration(1, 3, 6, 12 and 48 h), the proliferation and apoptosis of HUVECs were tested by CCK-8 assay and Annexin-V/PI double staining, respectively. Western blotting was adopted to determine the protein levels of Beclin-1, Hsc70 and Lamp-2. Finally, the time regulation characteristics of the 2 autophagy pathways before and after macroautophagy inhibition were analyzed. Results Under OX-LDL induced oxidative stress, the ROS level was elevated with the increase of OX-LDL concentration, and 50 μg/mL was finally selected as the appropriate concentration for subsequent experiments. As compared with the control group, the levels of Caspase-3, Beclin-1, Hsc70 and Lamp-2 were up-regulated, while that of PCNA was down-regulated in the oxidative stress group(P<0.05). With the prolongation of treatment time, proliferation inhibition rate, overall apoptotic rate and Lamp-2 level were increased within 48 h, Hsc70 level was elevated after 6 h, and that of Beclin-1 was improved within 12 h in the oxidative stress group(P<0.05). In the macroautophagy inhibition-oxidative stress group, the expression levels of Caspase-3, Hsc70 and Lamp-2 were also up-regulated, that of PCNA down-regulated(P<0.05), those of Hsc70 and Lamp-2 were increased at the same time and that of Hsc70 was in an elevation after 3 h of treatment(P<0.05). Conclusion Macroautophagy and CMA are activated successively during the process of oxidative stress, and CMA can be enhanced after macroautophagy inhibition. The early activation contributes to attenuate oxidative stress-induced damage to endothelial cells.