SIRT1在脂多糖诱导的PC12细胞凋亡中的作用

Role of SIRT1 in apoptosis of lipopolysaccharide-induced PC12 cells

目的探讨沉默信息调控因子1（SIRT1）在脂多糖（LPS）诱导的PCI2细胞凋亡过程中的作用。方法PCI2细胞按不同培养方法分为6组：对照组、250μg／mL组、500μg／mL组、750μg/mL组、1000μg／mL组、1250μg／mL组；对照组常规培养，后5组添加相应浓度的LPS培养。24h后MTT法测量细胞存活率，同时确定LPS诱导PCI2细胞凋亡的适合浓度。再按选定的浓度培养细胞，并根据培养时间的不同分为6组：对照组、1／2h组、2h组、18h组、24h组、48h组，Hoechst染色及流式细胞仪测量细胞凋亡率。Westernblotting检测各组细胞中SIRTl的表达情况。结果Hoechst染色结果提示PCI2细胞经LPS培养1／2h后出现凋亡，表现为核固缩、核碎裂：18h开始凋亡小体增多，24h达到高峰，48h后又有所下降。流式细胞仪检测结果显示各实验组细胞凋亡率与对照组相比差异均有统计学意义沪〈0．05），凋亡率变化趋势与Hoechst染色法观察到的结果一致。Westernblotting结果显示对照组SIRT1表达量为1．84±0．04：1／2h时SIRTl蛋白表达减低至1．17±0．09；24h时表达降至最低，为0．62±0．03；48h组表达量有所回升，达到0．77±0．02；实验组各时间点组与对照组相比差异均有统计学意义俨〈0．05）。结论LPS可以诱导PCI2细胞凋亡，SIRT1在此过程中的表达受到抑制；推测SIRT1在LPS诱导PCI2细胞凋亡的过程中起到一定的保护作用。

Objective To study the role of SIRT1 in apoptosis of PC12 neuronal cells induced by lipopolysaccharide （LPS）. Methods PC12 cells were cultured with different concentrations of LS （50μg/mL, 500 μg/mL, 750μg/mL, 1000μg/mL and 1250 μg/mL）, and some other PC12 cells were routinely cultured as controls. MTT assay was employed to identify the cell survival 24 h after the inducement, and accordingly, the suitable LPS concentration for subsequent experiments was determined based on MTT results. And then, cell apoptosis in the experimental groups under the suitable LPS concentration at different times （1/2, 2, 18, 24, and 48 h） and control group was noted by flow cytometry and Hoechst 33258 staining; Western blotting was used to detect the SIRT1 level in PC12 cells. Results Hoechst 33258 staining indicated that a few apoptotic bodies were noted 1/2 h after inducement, expressing as karyopyknosis and karyorrhexis; apoptotic bodies began to increase 18 h after inducement, reaching their peak level 24 h after inducement; and a decreased trend was observed 48 h after inducement. Flow cytometry indicated that significantly higher apoptosis rate at each time point was noted as compared with that in the control group （P〈0.05）; and Hoechst 33258 staining showed the same result. Western blotting revealed that the SIRT1 expression was （1.84±0.04） in the control group, decreasing to （1.17±0.09） 1/2 h after the inducement, and reaching the lowest level （0.62±0.03） 24 h after the inducement; and then, the expression was increased to （0.77±0.02） 48 h after the inducement;significant difference on the expression at each time point was noted as compared with that in the control group （P〈0.05）. Conclusion LPS can induce PC12 cell apoptosis and SIRT1 protein expression is inhibited, indicating that S1RT1 may take part in the apoptosis and play a protective role to PC12 cells.