脂多糖对大鼠胰岛素瘤细胞INS-1自噬和凋亡的影响

Effect of iipopolysaccharide on the autophagy and apoptosis in INS-1 rat insulinoma cells

目的研究脂多糖（LPS）对大鼠胰岛素瘤细胞INS-1自噬和凋亡的影响及其可能机制。方法取处于对数期生长的INS-1细胞用于实验，分别用0．1、1．0、10．0mg／LLPS处理细胞，24h后用磺酰罗丹明B（SRB）染色法检测LPS对INS-1细胞增殖的影响，用二氯荧光素双醋酸盐（DCFH-DA）作为荧光探针检测细胞内活性氧的水平变化，应用Western blotting法检测细胞自噬体膜上自噬标志分子微管相关蛋白1的轻链3-Ⅱ（Map1LC3-Ⅱ）和凋亡标志蛋白聚腺苷二磷酸-核糖多聚酶（PARP）切割带的变化。将INS-1细胞的自噬必需基因7（A增7）通过siRNA介导的RNA干扰手段进行沉默，然后以1．0mg／LLPS处理细胞，24h后应用Western blotting检测细胞中Atg7、Map1LC3-Ⅱ和PARP切割带的水平。先用400μmoL／L的活性氧清除剂N-乙酰半胱氨酸（NAC）处理INS-1细胞，1h后加入10．0mg／L的LPS，24h后应用Western blotting法检测细胞Map1LC3-Ⅱ蛋白和PARP切割蛋白的水平。组间数据比较采用方差分析和t检验。结果与对照组（0．755±0．030）比较，0．1、1．0、10．0mg／LLPS组INS-1存活率降低，差异有统计学意义（分别为0．658±0．042、0．658±0．015、0．634±0．029，F=30．98，P〈0．01）；与对照组（0．447±0．012）相比，0．1、1．0、10．0mg／LLPS组Map1LC3-Ⅱ蛋白水平增加，差异有统计学意义（分别为0．992±0．030、0．949±0．020、0．982±0．013，F=525．79，P〈0．01）；与对照组（0．055±0．001）相比，0．1、1．0、10．0mg／LLPS组PARP切割蛋白水平增加，差异有统计学意义（分别为0．313±0．007、0．390±0．011、0．500±0．033，F=327．09，均P〈0．01）；与对照组比较，10mg／LLPS组活性氧产生明显增多。与对照组比较，转染Atg7siRNA组Atg7、Map1LC3-Ⅱ和PARP切割蛋白水平均显著下降，差异均有统计学意义（t=156．069、123．154、103．246，均P〈0．01）。与LPS10．0mg／L组比较，LPS10．0mg／L＋NAC组Map1LC3-Ⅱ和PARP切割蛋白水平均显著下降，差异均有统计学意义（t=66．37、26．84，均P〈0．01）。结论LPS可诱导INS-1细胞的自噬和凋亡，且其所诱导的凋亡依赖于自噬的发生；活性氧参与了LPS诱导的INS-1细胞的自噬和凋亡。

Objective To investigate the effects of lipopolysaccharide （LPS） on autophagy and apoptosis of rat insulinoma INS-1 cells and the underlying mechanisms. Methods INS-1 cells at log growth phase were treated with 0. 1, 1.0 or 10. 0 mg/L LPS for 24 h, then sulforhodamine B （ SRB ） assay was carried out to evaluate the effect of LPS on cell viability, and reactive oxygen species （ROS） were detected by dichlorofluorescein-diacetate （DCFH-DA） fluoro-probe. The autophagy marker microtubule-associated protein 1 light chain 3 （ Map1 LC3-Ⅱ ） on the membrane of autophagosomes and the apoptosis marker cleaved poly ADP ribose polymerase （PARP） were detected by Western blotting analysis. The autophagy essential gene Atg7 was silenced via siRNA-mediated RNAi method, then cells were treated with 1.0 mg/L LPS for another 24 h. Atg7, Map1LC3-Ⅱ and cleaved PARP were detected by Western blotting analysis. INS-1 ceils were treated with 400 μmol/L N-acetylcysteine （ NAC）. And 1 h later the cells were treated with 10. 0 mg/L LPS for another 24 h. Map1LC3-Ⅱ and cleaved PARP were detected with Western blotting methods. Differences between groups were calculated with analysis of variance and t test. Results Compared with the control group （ 0. 755 ± 0. 030 ） , the cell viabilities significantly decreased in 0. 1, 1.0 and 10. 0 mg/L LPS groups （0. 658 ±0. 042, 0. 658 ± 0. 015, 0. 634 ± 0. 029, respectively, F = 30. 98, P 〈 0. 01 ） . Compared with the control group （ 0. 447 ± 0. 012 ）, the protein levels of Map1 LC3- Ⅱ significantly increased in 0. 1, 1.0 and 10. 0 mg/L LPS groups （0. 992 ±0. 030, 0. 949 ±0. 020, 0. 982 ± 0.013, respectively, F =525.79, P 〈0.01）. Compared with the control group （0.055 ±0.001）, the levels of cleaved PARP significantly increased in 0. 1, 1.0 and 10. 0 mg/L LPS groups （0. 313 ± 0. 007, 0. 390 ±0.011,0. 500 ±0.033,respectively, F =327.093, P 〈0.01）. A remarkable increase of ROS was detected in INS-1 cells treated with 10. 0 mg/L LPS for 24 h. Compared with the control, the protein levels of Atg7, Map1LC3-1/and cleaved PARP significantly decreased in Atg7 siRNA transfection group （t = 156. 069, 123. 154, 103. 246, all P 〈0. 01）. Compared with LPS 10. 0 mg/L group,the protein level of Map1LC3-Ⅱ and cleaved PARP significantly decreased in 10. 0 mg/L LPS ± NAC group （ t = 66. 37, 26. 84, P 〈 0. 01 ）. Conclusions LPS induces autophagy and apoptosis in INS-1 cells, and autophagy promotes apoptosis herein. Reactive oxygen species are involved in autophagy and apoptosis in INS-1 cells induced by LPS.