Toll样受体4介导的自噬减轻糖基化高密度脂蛋白诱导的血管内皮细胞凋亡

目的:探讨自噬对糖基化高密度脂蛋白(glycosylated high-density lipoprotein,gly-HDL)所致的血管内皮细胞凋亡的影响及其分子机制。方法:体外培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),分别与100 mg/L HDL和不同浓度(25、50和100 mg/L)gly-HDL共同孵育24 h;另再培养HUVECs给予1μmol/L自噬诱导剂雷帕霉素或2 mmol/L自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)预处理1 h,或5 mg/L抗Toll样受体4(Toll-like receptor 4,TLR4)单克隆中和抗体预处理30 min,再与gly-HDL(100 mg/L)共同孵育24 h。采用MTT法检测细胞活力,Annexin V-FITC/PI双染法检测细胞凋亡情况,试剂盒测定培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,采用Western blot技术检测自噬标志分子beclin-1和微管相关蛋白1轻链3-Ⅱ(microtubule-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ)、内质网应激凋亡途径关键分子caspase-12及TLR4的表达变化,采用激光共聚焦显微镜观测细胞内LC3的变化。结果:经gly-HDL处理的HUVECs活力下降,LDH漏出和细胞凋亡显著增加(P<0.01),且caspase-12被激活(P<0.05);雷帕霉素预处理HUVECs后,gly-HDL对细胞的损伤作用和对caspase-12的活化作用减弱(P<0.05);而3-MA预处理HUVECs后,gly-HDL对细胞的损伤作用和对caspase-12的活化作用则进一步加强(P<0.05)。gly-HDL显著上调TLR4的表达,并触发自噬反应,表现为beclin-1和LC3-Ⅱ表达上调及LC3显著颗粒化,且呈浓度依赖性(P<0.05);而抗TLR4单克隆中和抗体预处理可显著抑制gly-HDL所诱导的beclin-1上调和LC3颗粒化(P<0.01)。结论:TLR4介导gly-HDL对HUVECs自噬的诱导作用,而一定程度的自噬可通过抑制caspase-12活化减轻gly-HDL所诱导的HUVECs凋亡。

Toll-like receptor 4-mediated autophagy attenuates glycosylated highdensity lipoprotein-induced apoptosis of vascular endothelial cells

AIM:To investigate the effect of autophagy on glycosylated high-density lipoprotein(gly-HDL)-induced vascular endothelial cell apoptosis and the underlying molecular mechanisms.METHODS:Human umbilical vein endothelial cells(HUVECs)were treated with gly-HDL(25,50 and 100 mg/L)and HDL(100 mg/L)for 24 h.In addition,the HUVECs were pretreated with 1μmol/L rapamycin or 2 mmol/L 3-methyladenine(3-MA)for 1 h,or 5 mg/L anti-Toll-like receptor 4(TLR4)monoclonal antibody for 30 min,and then treated with gly-HDL(100 mg/L)for 24 h.MTT assay and Annexin V-FITC/PI double staining were used to analyze the viability and apoptosis,respectively.The activity of lactate dehydrogenase(LDH)in culture medium was measured.Western blot was used to determine the expression changes of beclin-1,microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),caspase-12 and TLR4.Laser confocal microscopy was used to observe the changes of LC3 in cells.RESULTS:Treatment of HUVECs with gly-HDL resulted in dramatic decrease of viability,significant elevation of LDH leakage and apoptosis,as well as activation of caspase-12(P<0.05),which were attenuated by autophagy inducer rapamycin and aggravated by autophagy inhibitor 3-MA(P<0.05).Moreover,gly-HDL up-regulated TLR4 expression and induced autophagy in HUVECs as evaluated by up-regulation of beclin-1,LC3-Ⅱ and frequent granulation of LC3 in a dose-dependent manner(P<0.05).However,anti-TLR4 neutralizing antibody significantly inhibited up-regulation of gly-HDL-induced beclin-1 and frequent granulation of LC3(P<0.01).CONCLUSION:TLR4 mediates gly-HDL-induced autophagy in HUVECs,and moderates activation of autophagy,which may protect HUVECs from gly-HDL-induced apoptosis by inhibiting caspase-12 activation.