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目的:研究蜂胶醇取物(ethanol extract of propolis,EEP)对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的巨噬细胞凋亡的抑制作用,并探讨可能的分子机制。方法:体外培养RAW264.7巨噬细胞,给予EEP(7.5、15和30 mg/L)、4-苯丁酸(4-phenylbutyric acid,PBA;5 mmol/L)或二亚苯基碘鎓(diphenyleneiodonium,DPI;5μmol/L)预处理1 h,再加入ox-LDL(100 mg/L)或衣霉素(tunicamycin,TM;4 mg/L)继续培养24 h。分别采用MTT法和Annexin V-FITC双染法检测细胞活力和凋亡情况;试剂盒测定细胞内超氧化物歧化酶(superoxide dismutase,SOD)活性、活性氧簇(reactive oxygen species,ROS)和丙二醛(malondialdehyde,MDA)的水平。采用免疫印迹技术检测内质网应激(endoplasmic reticulum stress,ERS)凋亡途径关键蛋白caspase-12的表达变化。结果:与ERS抑制剂PBA相似,EEP呈剂量依赖性减轻ox-LDL所致的巨噬细胞损伤,表现为细胞活力增加(P<0.01),凋亡率降低(P<0.05),且可抑制ERS诱导剂TM所引起的巨噬细胞活力下降和凋亡(P<0.05);与氧化应激抑制剂DPI相似,EEP可抑制ox-LDL诱导的氧化应激反应,表现为ROS和MDA生成减少(P<0.01),SOD活性增加(P<0.05);EEP显著抑制ox-LDL和TM所诱导的caspase-12活化(P<0.05);与ox-LDL组比较,PBA和DPI预处理组caspase-12活性也受到明显抑制(P<0.01)。结论:EEP可减轻ox-LDL所诱导的RAW264.7巨噬细胞凋亡,其机制可能与抑制氧化应激和caspase-12活化有关。
OBJECTIVE: To study the inhibitory effect of ethanol extract of propolis (EEP) on the apoptosis of macrophages induced by oxidized low-density lipoprotein (ox-LDL) and to explore the possible molecular mechanisms. METHODS: RAW264.7 macrophages were cultured in vitro and treated with EEP (7.5,15 and 30 mg / L), 4-phenylbutyric acid (PBA; 5 mmol / L) or diphenyleneiodonium diphenyleneiodonium (DPI) and 5μmol / L pretreatment for 1 h, followed by incubation with ox-LDL (100 mg / L) or tunicamycin (TM) at 4 mg / L for 24 h. Cell viability and apoptosis were detected by MTT assay and Annexin V-FITC double staining respectively. The activity of superoxide dismutase (SOD), reactive oxygen species (ROS) and C The level of malondialdehyde (MDA). Western blotting was used to detect the expression of caspase-12, a key protein in apoptosis pathway of endoplasmic reticulum stress (ERS). Results: Similar to ERS inhibitor PBA, EEP reduced the ox-LDL-induced macrophage injury in a dose-dependent manner, with increased viability and apoptotic rate (P <0.01) (P <0.05). Similar to the DPI, EEP inhibited ox-LDL-induced oxidative stress and showed decreased ROS and MDA production (P <0.01), while the activity of SOD increased (P <0.05). EEP significantly inhibited the activation of caspase-12 induced by ox-LDL and TM (P <0.05) -12 activity was also significantly inhibited (P <0.01). Conclusion: EEP can reduce the apoptosis of RAW264.7 macrophages induced by ox-LDL, which may be related to the inhibition of oxidative stress and the activation of caspase-12.