二氢杨梅素调控自噬抑制氧化低密度脂蛋白诱导人脐静脉内皮细胞损伤的研究

Dihydromyricetin protects oxidized low-density lipoproteininduced damage by inducing autophagy in human umbilical vein endothelial cells

摘要:

目的观察二氢杨梅素（DMY）对氧化低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞（HUVECs）损伤的影响，并探讨自噬在其中的作用。方法DMY（0.01、0.10、1.00和10.00 μmol/L）预处理HUVECs 2 h，用100.0 mg/L ox-LDL 继续培养细胞24 h。以辛伐他汀作为阳性对照组。采用噻唑蓝法检测细胞活力，Hoechst33258染色观察细胞核形态，透射电镜观察自噬体，Western blot检测自噬相关蛋白微管相关蛋白1轻链3（LC3）、Beclin-1和p62/SQSTM1的表达，观察自噬抑制剂对DMY作用的影响。结果与对照组比较，ox-LDL组细胞的存活率降低（p <0.05），细胞核呈集中高强度蓝色荧光，固缩致密浓染或碎块状致密浓染，颜色发白，细胞核较小，形态不规则，自噬体和自噬溶酶体数量增加；Beclin-1、LC3-Ⅱ的表达和LC3-Ⅱ/LC3-Ⅰ比值上调，而p62的表达下调（p <0.05）。与ox-LDL组比较，0.1、1.0和10.0μmol/L DMY预处理组细胞存活率均升高；细胞核荧光强度降低，核固缩致密浓染和碎块状致密浓染减少，形态较规则；1.0 μmol/L DMY 预处理组的自噬体和自噬溶酶体数量增加；Beclin-1、LC3-Ⅱ的表达和LC3-Ⅱ/LC3-Ⅰ比值上调，而p62 的表达下调（p <0.05）。自噬抑制剂3-MA 部分抵消DMY 抑制ox-LDL诱导的HUVECs 存活率降低（p <0.05）。结论DMY 能抑制ox-LDL诱导的HUVECs 损伤，其机制与诱导自噬有关。

Abstract:

Objective To investigate the effect of Dihydromyricetin (DMY) on the damage induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and to explore the role of autophagy in this effect. Methods After HUVECs were pretreated with DMY (0.01, 0.1, 1.0 and 10.0 μmol/L) for 2 h, the cells were incubated with ox-LDL (100.0 mg/L) for 24 h. Simvastatin was made as a positive control. MTT was used to detect cell viability. Nuclear morphology was observed by Hoechst 33258 staining. Cell ultrastructures and autophagosomes were observed by transmission electron microscope. The expressions of Beclin-1, light chain-3 of microtubule-associated protein (LC3) and the autophagy substrates p62/SQSTM1 in cells were determined by Western blot. Results Compared with the control group, the cell survival rate was singificantly decreased (p < 0.05), the nuclei were smaller in the ox-LDL group. The concentrated high-intensity blue fluorescence in nuclei, nuclear condensation and dense dyeing, or nuclear chunky and dense dyeing were observed in the ox-LDL group. Compared with the control group, the number of autolysosomes was obviously increased, the expressions of Beclin-1 and LC3-Ⅱand the ratio of LC3-Ⅱ/LC3-Ⅰin the cells were singificantly increased and the expression of p62 in the cells was singificantly decreased in the ox-LDL groups (p < 0.05). Compared with the ox-LDL group, the cell survival rate was singificantly increased (p < 0.05), the fluorescence intensity of nuclei was obviously decreased, nuclear condensation and dense dyeing or nuclear chunky and dense dyeing were decreased, the nuclei were regular in the DMY (0.1, 1.0 and 10.0 μmol/L) pretreatment groups. Compared with the ox-LDL group, the number of autolysosomes was obviously increased, the expressions of Beclin-1 and LC3-Ⅱand the ratio of LC3-Ⅱ/LC3-Ⅰin the cells were singificantly increased while the expression of p62 in the cells was singificantly decreased in the DMY (1.0 μⅡmol/L) pretreatment group (p < 0.05). Autophagy inhibitor 3-MA partly abolished the effect of DMY on the cell survival rate (p < 0.05). Conclusions DMY protects ox-LDL-induced damage in HUVECs, the mechanism is associated with induction of autophagy.