载脂蛋白A-I模拟肽D4F通过抑制caspase-12减轻氧化低密度脂蛋白诱导的巨噬细胞凋亡  

Apolipoprotein A-I mimetic peptide D4F protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting caspase-12

作　　者：田华[1] 李严严[1] 丁明德[2] 杨娜娜[1] 焦鹏[1] 桑慧[1,3] 方永奇[3] 姚树桐[1,3] 秦树存[1] 

机构地区：[1]泰山医学院动脉粥样硬化研究所,山东省高校动脉粥样硬化重点实验室,山东泰安271000 [2]泰山医学院附属医院,山东泰安271000 [3]泰山医学院基础医学院,山东泰安271000

出　　处：《中国病理生理杂志》2015年第10期1750-1755,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81202949;No.81370381);山东省自然科学基金联合专项(No.ZR2014HL013);山东省医药卫生科技发展计划(No.2013WSB31009)

摘　　要：目的:探讨载脂蛋白A-I(apolipoprotein A-I,Apo A-I)模拟肽D4F对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的巨噬细胞凋亡和内质网应激(endoplasmic reticulum stress,ERS)凋亡途径关键分子caspase-12的影响,并阐明其可能的分子机制。方法:体外培养RAW264.7巨噬细胞,给予12.5、25和50 mg/L D4F、5 mmol/L ERS抑制剂4-苯丁酸(4-phenylbutyric acid,PBA)或5μmol/L二亚苯基碘鎓(diphenyleneiodonium,DPI)预处理1 h后,再加入100 mg/L ox-LDL或4 mg/L ERS诱导剂衣霉素(tunicamycin,TM)继续培养24 h。MTT法检测细胞活力;TUNEL法检测细胞凋亡情况;试剂盒测定细胞内丙二醛(malondialdehyde,MDA)和活性氧(reactive oxygen species,ROS)水平,以及超氧化物歧化酶(superoxide dismutase,SOD)和烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶活性;Western blot法检测caspase-12的表达变化。结果:与ERS抑制剂PBA相似,D4F可抑制ox-LDL或TM所致的巨噬细胞活力降低和凋亡,且呈浓度依赖性(P<0.05)。与氧化应激抑制剂DPI相似,D4F显著抑制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活性增加以及NADPH氧化酶活性降低(P<0.05);与PBA和DPI相似,D4F可减轻ox-LDL诱导的巨噬细胞caspase-12活化,且呈浓度依赖性(P<0.05);另外,D4F还可抑制TM诱导的caspase-12活化(P<0.05)。结论:D4F能够抑制ox-LDL诱导的巨噬细胞凋亡,其机制至少部分是通过减轻氧化应激继而抑制caspase-12活化实现的。AIM： To investigate the effect of D4F, an apolipoprotein A-I mimetic peptide, on oxidized low- density lipoprotein （ox-LDL）-induced macrophage apoptosis and activation of caspase-12, a key molecule in endoplasmic reticulum stress （ERS）-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms. METHODS： RAW264. 7 macrophages were pretreated with D4F （12. 5,25 and 50 mg/L）, 4-phenylbutyric acid （PBA, 5 mmol/L） or diphenyleneiodonium （DPI, 5 μmol/L） for 1 h and then treated with ox-LDL （100 mg/L） or tunicamycin （TM, 4 mg/L） for 24 h. The cell viability and apoptosis were determined by MTT assay and TUNEL detection, respective- ly. The levels of malondialdehyde （MDA） and reactive oxygen species （ROS） in the cells and the activities of superoxide dismutase （SOD） and nicotinamide adenine dinucleotide phosphate （NADPH） oxidase were determined. The protein level of caspase-12 was examined by Western blot analysis. RESULTS： Similar to the ERS inhibitor PBA, D4F protectedRAW264. 7 macrophages from ox-LDL or TM （ an ERS inducer） -induced decrease in the viability and increase in apoptotic rate in a dose-dependent manner. Like DPI （ an oxidative stress inhibitor） , D4F significantly inhibited ox-LDL-induced ox- idative stress, as expressed by the decreased generation of ROS and MDA （P 〈 0. 01 ）, the increased activity of SOD and the decreased activity of NADPH oxidase （ P 〈 0.05 ）. Moreover, similar to PBA and DPI, D4F significantly suppressed ox-LDL-induced activation of caspase-12 in a concentration-dependent manner （P 〈 0. 05）. Furthermore, D4F also inhibi- ted the caspase-12 activation induced by TM （ P 〈 0.05 ）. CONCLUSION ： D4F inhibits macrophage apoptosis induced by ox-LDL, and the mechanism is at least partially by reducing oxidative stress and inhibiting the activation of caspase-12.

关 键 词：载脂蛋白A-I模拟肽D4F CASPASE-12 氧化低密度脂蛋白 巨噬细胞 细胞凋亡 

分 类 号：R363.2[医药卫生—病理学] R332[医药卫生—基础医学]