半胱天冬蛋白酶-3在高迁移率族蛋白B1诱导小鼠巨噬细胞凋亡中的作用

Role of cysteinyl aspartate-specific protease 3 signaling in high mobility group box-1 protein-induced apoptosis of peritoneal macrophages in mice

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.