CAJ | 학술논문

目的：探讨载脂蛋白A－I（apolipoproteinA－I，ApoA－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（ reac－tive 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（nic－otinamide 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。
목적：탐토재지단백A－I（apolipoproteinA－I，ApoA－I）모의태D4F대양화저밀도지단백（oxidized low－density lipoprotein，ox－LDL）유도적거서세포조망화내질망응격（ endoplasmic reticulum stress，ERS）조망도경관건분자caspase－12적영향，병천명기가능적분자궤제。방법：체외배양RAW264．7거서세포，급여12．5、25화50 mg／L D4F、5 mmol／L ERS억제제4－분정산（4－phenylbutyric acid，PBA）혹5μmol／L이아분기전옹（ diphenyleneiodonium， DPI）예처리1 h후，재가입100 mg／L ox－LDL혹4 mg／L ERS유도제의매소（ tunicamycin，TM）계속배양24 h。 MTT법검측세포활력；TUNEL법검측세포조망정황；시제합측정세포내병이철（ malondialdehyde，MDA）화활성양（ reac－tive oxygen species，ROS）수평，이급초양화물기화매（superoxide dismutase，SOD）화연선알선표령이핵감산린산（nic－otinamide adenine dinucleotide phosphate，NADPH）양화매활성；Western blot법검측caspase－12적표체변화。결과：여ERS억제제PBA상사，D4F가억제ox－LDL혹TM소치적거서세포활력강저화조망，차정농도의뢰성（P＜0．05）。여양화응격억제제DPI상사，D4F현저억제ox－LDL유도적양화응격반응，표현위ROS화MDA생성감소（ P＜0．01）、SOD활성증가이급NADPH양화매활성강저（P＜0．05）；여PBA화DPI상사，D4F가감경ox－LDL유도적거서세포caspase－12활화，차정농도의뢰성（P＜0．05）；령외，D4F환가억제TM유도적caspase－12활화（P＜0．05）。결론：D4F능구억제ox－LDL유도적거서세포조망，기궤제지소부분시통과감경양화응격계이억제caspase－12활화실현적。
[ABSTRACT]AIM:ToinvestigatetheeffectofD4F,anapolipoproteinA-Imimeticpeptide,onoxidizedlow-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 protected RAW264.7 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.