摘要目的:评价抑制Ras相关的C3肉毒素底物1(ras-related C3 botulinum toxin substrate 1, Rac1）表达对高糖诱发神经元损伤的影响。方法:对数生长期PC12细胞接种于6孔板，采用随机数字表法将细胞分为4组（每组3孔）：正常浓度葡萄糖组（C组）、高浓度葡萄糖组（HG组）、高浓度葡萄糖+慢病毒抑制Rac1组（HG+shRac1组）、高浓度葡萄糖+慢病毒阴性对照组（HG+NC组）。C组以葡萄糖浓度为4.5 g/L的DMEM完全培养基培养PC12细胞24 h, HG组以葡萄糖浓度为45 g/L的DMEM完全培养基培养PC12细胞24 h, HG+shRac1组以葡萄糖浓度为45 g/L的DMEM完全培养基培养用Rac1 shRNA慢病毒载体转染的PC12细胞24 h，HG+NC组以葡萄糖浓度为45 g/L的DMEM完全培养基培养用阴性慢病毒载体转染的PC12细胞24 h。Western blot法检测微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3, LC3）Ⅰ、LC3Ⅱ、Bcl-2/腺病毒E1B相互作用蛋白3 （Bcl-2/adenovirus E1B protein-interacting protein 3, BNIP3）和p62蛋白水平，并计算LC3Ⅱ/LC3Ⅰ；CCK-8法检测细胞活力；流式细胞术检测细胞凋亡率；二氢溴化乙啶法检测细胞活性氧（reactive oxygen species, ROS）水平；荧光定量PCR法检测自噬相关基因7 (autophagy-related gene 7, Atg7）、三磷酸腺苷酶6 （adenosinetriphosphatase 6, ATPase6）和60S核糖体蛋白L13 （60S ribosomal protein L13, Rpl13）相对表达量，并计算ATPase6/Rpl13。结果:与C组比较，HG组和HG+NC组ROS水平和细胞凋亡率升高（ P<0.05），细胞活力下降（ P<0.05）；与HG+NC组比较，HG+shRac1组ROS水平和细胞凋亡率降低（ P< 0.05），细胞活力升高（ P<0.05），Atg7相对表达量和BNIP3蛋白水平升高（ P<0.05），p62蛋白水平降低（ P<0.05），LC3Ⅱ/LC3Ⅰ升高（ P<0.05），ATPase6/Rpl13降低（ P<0.05）。 结论:抑制Rac1可减轻高糖诱发的神经元损伤，其机制可能与增强线粒体自噬有关。

abstractsObjective:To evaluate the effect of inhibiting ras-related C3 botulinum toxin substrate 1 (Rac1) expression on high glucose-induced neurotoxicity.Methods:PC12 cells at the logarithmic growth phase were seeded in 6-well plates. According to the random number table method, they were divided into four groups ( n=3): a normal glucose group (group C), a high glucose group (group HG), a high glucose+lentivirus inhibiting Rac1 small hairpin RNA group (group HG+shRac1) and a high glucose+lentivirus negative control group (group HG+NC). PC12 cells in group C were cultured in DMEM with 4.5 g/L glucose for 24 h, the cells in group HG were cultured in DMEM with 45 g/L glucose for 24 h, the cells in group HG+shRac1 were cultured in DMEM medium with 45 g/L glucose and transfected with lentiviral vector expressing shRNA of Rac1 for 24 h, and the cells in group HG+NC were transfected with negative lentiviral vector in DMEM medium containing a glucose concentration of 45 g/L for 24 h. Then, the levels of microtubule-associated protein 1 light chain 3 (LC3)Ⅰ, LC3Ⅱ, Bcl-2/adenovirus E1B protein-interacting protein 3 (BNIP3) and p62 were detected by Western blot and the ratio of LC3Ⅱ/LC3Ⅰ was calculated. The cellular viability was detected by CCK8 assay. The apoptotic rate was measured by flow cytometry. The levels of cellular reactive oxygen species (ROS) was detected by dihydroethidium. Moreover, the relative expression of autophagy-related gene 7 (Atg7), adenosinetriphosphatase 6 (ATPase6) and 60S ribosomal protein L13 (Rpl13) were detected by real-time fluorescent polymerase chain reaction (PCR), and the ratio of ATPase6/Rpl13 was calculated. Results:Compared with group C, group HG and group HG+NC showed increases in ROS production and apoptotic rate ( P<0.05), and decreases in cellular viability ( P<0.05). Compared with group HG+NC, group HG+shRac1 presented decreases in ROS production and apoptotic rate ( P<0.05), and increases in cellular viability ( P<0.05), up-regulated Atg7 relative expression and BNIP3 protein levels ( P< 0.05), down-regulated p62 protein levels ( P<0.05), increased LC3 Ⅱ/LC3 Ⅰ ratio ( P<0.05) and decreased ATPase6/Rpl13 ratio ( P< 0.05). Conclusions:Inhibition of Rac1 can attenuate high glucose-induced neurotoxicity, which may be associated with enhanced mitophagy.