摘要
目的观察半胱天冬蛋白酶(Caspase)-3与核转录因子(NF).KB在高迁移率族蛋白B1(HMGBl)诱导小鼠腹腔巨噬细胞凋亡中的作用。方法分离培养小鼠腹腔巨噬细胞,随机分为对照组、HMGB1组与抑制剂组(HMGB1作用前1h加入Caspase-3抑制剂Z,DEVD-FMK)。采用Hoechst33342染色观察凋亡的形态学变化、流式细胞术检测巨噬细胞凋亡百分率的变化;应用原位荧光染色及流式细胞术检测Caspase-3活性的变化;同时应用酶联免疫吸附试验检测NF—KBp65蛋白的变化。结果HMGBl组巨噬细胞凋亡率为(38.21±4.85)%、抑制剂组为(16.47±3.91)%,均显著高于对照组(4.21±1.64)%(P〈0.01)。经Hoechst33342染色,荧光显微镜观察HMGBl组凋亡细胞明显增多;原位荧光染色观察到HMGB1组Caspase.3荧光强度明显增强,Caspase-3阳性细胞百分率(29.74±4.55)%显著高于抑制剂组(3.02±1.91)%与对照组(7.19±2.46)%(P〈0.01)。同时,HMGB1组、抑制剂组细胞核NF.KB活性较对照组显著降低(P〈0.05或P〈0.01)。结论Caspase-3和NF-KB是HMGB1诱导巨噬细胞凋亡的重要途径,caspase-3抑制剂可部分阻断HMGB1诱导的巨噬细胞凋亡。
Objective To investigate the potential role of cysteinyl aspartate-specific protease (Caspase) 3 and nuclear factor kappa B (NF-KB) signaling in high mobility group box-1 protein ( HMGB1 ) -induced apoptosis of peritoneal macrophages in mice. Methods Peritoneal macrophages from female BALB/c mice were randomly divided into 3 groups:control group, HMGB1 group and Caspase-3 inhibitor group ( 40 μmol/L Z-DEVD-FMK was added one h before exposure to 10 mg/L HMGB1 ). Apoptotic cells were stained with Annexin V-PE and 7-AAD and examined by flow eytometry, and nuclear features of apoptosis were examined by Hoechst33342. Activated Caspase-3 was measured by fluorescence microscopy and flow cyt0metry after staining with the in situ marker FITC-DEVD-FMK. Nuclear p65 activity was determined using the TransAM Transcription Factor DNA-binding ELISAs ( NF-KB p65 ). Results After stimulation with 10 mg/L HMGB1 for 24 h, the apoptosis rate of macrophages was ( 38. 21 ± 4.85) % in HMGB1 group and ( 16.47±3.91 ) % in Caspase-3 inhibitor group, respectively, which was significantly higher than that in controls [ (4.21±1.64 ) % , P 〈 0.01 ]. The percentage of apoptotic nuclei staining with Hoechst3342 in HMGB1 group was higher than that in the other groups. The Caspase-3 posi- tive macrophage was ( 29.74±4.55 ) % in HMGB1 group, which was significantly higher than that in Caspase-3 inhibitor group [ (3.02± 1.91 ) %, P 〈 0.01 ] and control group [ ( 7.19± 2.46 )%, P 〈 0.01 ]. As compared with control group,activities of NF-KB p65 in both HMGB1 and Caspase-3 inhibitor groups were markedly decreased ( P 〈 0.05 or P 〈 0.01 ). Conclusion HMGB1 can induce apoptosis of murine macrophages via activation of Caspase-3 and inhibition of NF-KB pathway. Treatment with Caspase- 3 inhibitor might partially block HMGBl-induced apoptosis.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2008年第10期1248-1250,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30672178)
国家杰出肖年科学基金资助项目(30125020)
国家重点基础研究发展规划项目(2005CB522602)
关键词
高迁移率族蛋白B1
脱噬作用
巨噬细胞
半胱天冬蛋白酶-3
核因子KB
High mobility group box-1 protein
Apoptosis
Macrophage
Cysteinyl aspartate-specific protease 3
Nuclear factor-kappa B