N-乙酰半胱氨酸对内质网应激介导的HepG2细胞凋亡的影响
[The effect of N-acetyl-L-cysteine on endoplasmic reticulum stress mediated apoptosis of HepG2 cells].
作者信息
Liu Yun-ye, Xie Qing, Wang Hui, Lin Lan-yi, Jiang Shan, Zhou Xia-qiu, Yu Hong, Guo Qing
机构信息
Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University Medical School, Shanghai 200025, China.
出版信息
Zhonghua Gan Zang Bing Za Zhi. 2008 Jul;16(7):524-7.
OBJECTIVE
To analyze the mechanisms of NAC on endoplasmic reticulum (ER) stress mediated cells apoptosis of HepG2 cells and to evaluate the potential role of NAC in the treatment of liver injury.
METHODS
HepG2 cells were treated with H2O2 to make a model of oxidative ER stress mediated apoptosis. To evaluate the apoptosis, various methods such as MTT, DNA ladder, Western blot and flow cytometry were used. Then the optimal dosage and incubation time of NAC intervention in apoptosis were ascertained, and the differences between induction and intervention of apoptosis, including the percentage of apoptosis, the expression of apoptotic protein (GRP78, Caspase-12, PARP) and the production of reactive oxygen species (ROS) were compared.
RESULTS
The activity of the cells decreased by H2O2 (0, 1, 3, 5 mmol/L) treatments in a dose-dependent manner. The ratio of apoptotic cells increased (0.7%+/-0.5%, 26.4%+/-1.8%, 29.7%+/-1.2% and 51.2%+/-9.4%, respectively) as did the production of ROS (14.0%+/-0.5%, 95.2%+/-0.1%, 97.5%+/-0.2% and 98.3%+/-0.2%, respectively). The HepG2 cells showed typical morphologic change of ER stress 6 hr after they were treated with 3 mmol/L H2O2. ER stress mediated-apoptosis was confirmed by Western blot. NAC (10 mmol/L and 20 mmol/L) protected cells from apoptosis. Typical features of ER stress apoptosis were seen accompanied by diminishing the ratio of apoptotic cells from 29.7%+/-1.2% to 23.3%+/-4.7% and 14.3%+/-1.2%. The production of ROS also decreased from 97.5%+/-0.2% to 52.2%+/-0.8% and 51.2%+/-2.9%. The effect was related to the concentration: 20 mmol/L NAC was more effective than 10 mmol/L.
CONCLUSIONS
As an oxidizing agent, H2O2 may induce ROS in cells and induce oxidative stress, causing ER stress and apoptosis. NAC can inhibit the procession of ROS directly and prevent injuries to the hepatocytes.
目的
分析N-乙酰半胱氨酸（NAC）对内质网（ER）应激介导的HepG2细胞凋亡的作用机制，并评估NAC在肝损伤治疗中的潜在作用。
方法
用H2O2处理HepG2细胞，建立氧化型ER应激介导的细胞凋亡模型。采用MTT法、DNA梯状条带法、蛋白质免疫印迹法和流式细胞术等多种方法评估细胞凋亡情况。确定NAC干预细胞凋亡的最佳剂量和孵育时间，并比较诱导凋亡和干预凋亡之间的差异，包括凋亡率、凋亡蛋白（GRP78、Caspase-12、PARP）表达以及活性氧（ROS）生成。
结果
用H2O2（0、1、3、5 mmol/L）处理后，细胞活性呈剂量依赖性降低。凋亡细胞比例增加（分别为0.7%±0.5%、26.4%±1.8%、29.7%±1.2%和51.2%±9.4%），ROS生成也增加（分别为14.0%±0.5%、95.2%±0.1%、97.5%±0.2%和98.3%±0.2%）。用3 mmol/L H2O2处理HepG2细胞6小时后，细胞呈现典型的ER应激形态学变化。蛋白质免疫印迹法证实存在ER应激介导的细胞凋亡。NAC（10 mmol/L和20 mmol/L）可保护细胞免于凋亡。ER应激凋亡的典型特征出现，同时凋亡细胞比例从29.7%±1.2%降至23.3%±4.7%和14.3%±1.2%。ROS生成也从97.5%±0.2%降至52.2%±0.8%和51.2%±2.9%。该作用与浓度有关：20 mmol/L NAC比10 mmol/L更有效。
结论
作为一种氧化剂，H2O2可诱导细胞内ROS生成并引发氧化应激，导致ER应激和细胞凋亡。NAC可直接抑制ROS的产生，防止肝细胞损伤。
Zotero 插件