Li Tian-Ti, Li Zhao-Hui, Zhuang Xu-Ying, Fu Yu-Cai
Department of Forensic Medicine, Zhongshan Medical College of SUN Yat-sen University, Guangzhou 510080, China.
Zhonghua Yi Xue Za Zhi. 2011 Feb 1;91(5):350-8.
To study the regulation effects of CR (caloric restriction) and SIRT3 in the H2O2-induced oxidative stress injury of PC12 cell.
The cells were divided into four groups: H2O2, H2O2 + CR, CR and control (high glucose). For control and H2O2 group, cells were cultured in DMEM containing 0.45% glucose; for group in CR condition, cells were treated with the medium containing 0.1% glucose. For groups with H2O2, the H2O2 was diluted in EMEM medium to obtain the final concentration containing 60 µmol/L. Viability of PC12 cells were measured by MTT assay. The medium was refreshed with different concentration of H2O2 (from 10 to 120 µmol/L). The absorbance of the samples was measured at 492 nm using a microtiter plate reader. We detected TUNEL-positive cells using the In Situ Cell Apoptosis Detection kit pretreated with CR and H2O2. Immunofluorescence double staining detected the expression and localization of SIRT3. RT-PCR and Western-blot methods detected the expression of SIRT3, Caspase-3.
After pretreating with 60 µmol/L H2O2 for 6 h, the viability of PC12 cells in H2O2 group (74.01 ± 2.21)% retained above 70%, and have statistical significance contrasted with control group (P < 0.05); After pretreating with 120 µmol/L H2O2, the viability of PC12 cells declined significantly (38.22 ± 3.34)%. So 60 µmol/L H2O2 is our experiment concentration. The viability of H2O2 group (74.01 ± 2.21)% was much lower than CR + H2O2 group (97.26 ± 1.92)% (P < 0.05). After pretreating with H2O2, TUNEL staining showed the apoptosis cells of CR + H2O2 group decreased significantly contrasted with H2O2 group. The immunofluorescence double staining results showed that SIRT3 was a mitochondria protein. Western-blot showed the expression of SIRT3 in CR group (6857 ± 157) (P < 0.05) increased and decreased in H2O2 group (3786 ± 160) (P < 0.05) contrasted with control group (5256 ± 143). The expression of SIRT3 in CR + H2O2 group (5056 ± 121) (P < 0.05) increased contrasted with H2O2 group (3786 ± 160). We also detected that Caspase-3 in H2O2 group (8499 ± 426) (P < 0.001) was much higher than control group than (5342 ± 420), but in the CR + H2O2 group (5750 ± 438) the expression of Caspase-3 was much lower than H2O2 group (8499 ± 426) (P < 0.001). RT-PCR also showed that the expression of SIRT3 in CR group (7214 ± 148) increased and decreased in H2O2 group (4807 ± 143) (P < 0.05) contrasted with control group (6204 ± 134). The expression of SIRT3 in CR + H2O2 group (6195 ± 166) increased contrasted with H2O2 group (4807 ± 143) (P < 0.05).
CR causes anti-oxidative injury and has apoptotic effects in PC12 cell. It up-regulates the expression of SIRT3 and the effects of CR-SIRT3 can prevent PC12 cell from H2O2-induced apoptosis. And SIRT3 may be a novel molecule of regulating target in the delay of neuronal senescence.
研究热量限制（CR）和SIRT3对H2O2诱导的PC12细胞氧化应激损伤的调控作用。
将细胞分为四组：H2O2组、H2O2 + CR组、CR组和对照组（高糖）。对照组和H2O2组细胞在含0.45%葡萄糖的DMEM培养基中培养；CR条件组细胞用含0.1%葡萄糖的培养基处理。对于H2O2处理组，将H2O2在EMEM培养基中稀释以获得终浓度为60 μmol/L。采用MTT法检测PC12细胞活力。用不同浓度的H2O2（10至120 μmol/L）更换培养基。使用酶标仪在492 nm处测量样品的吸光度。我们使用经CR和H2O2预处理的原位细胞凋亡检测试剂盒检测TUNEL阳性细胞。免疫荧光双染检测SIRT3的表达和定位。采用RT-PCR和Western-blot方法检测SIRT3、Caspase-3的表达。
用60 μmol/L H2O2预处理6小时后，H2O2组PC12细胞活力（74.01±2.21）%保持在70%以上，与对照组相比有统计学意义（P<0.05）；用120 μmol/L H2O2预处理后，PC12细胞活力显著下降（38.22±3.34）%。所以60 μmol/L H2O2是我们的实验浓度。H2O2组细胞活力（74.01±2.21）%远低于CR + H2O2组（97.26±1.92）%（P<0.05）。用H2O2预处理后，TUNEL染色显示CR + H2O2组凋亡细胞较H2O2组显著减少。免疫荧光双染结果显示SIRT3是一种线粒体蛋白。Western-blot显示，与对照组（5256±143）相比，CR组SIRT3表达（6857±157）升高（P<0.05），H2O2组降低（3786±160）（P<0.05）。与H2O2组（3786±160）相比，CR + H2O2组SIRT3表达（5056±121）升高（P<0.05）。我们还检测到，H2O2组Caspase-3（8499±426）（P<0.001）远高于对照组（5342±420），但CR + H2O2组Caspase-3表达（5750±438）远低于H2O2组（8499±426）（P<0.001）。RT-PCR也显示，与对照组（6204±134）相比，CR组SIRT3表达（7214±148）升高，H
