N-乙酰半胱氨酸对HT22细胞辐射诱导氧化应激及增殖和凋亡的影响

为探讨N-乙酰-L-半胱氨酸(N-acetylcysteine, NAC)对辐射相关氧化应激和海马神经元HT22细胞的增殖及凋亡的影响。首先选用不同剂量(0、2、4、6、8、10、12 Gy)的X射线分别照射HT22细胞,筛选出最佳照射剂量(10 Gy),然后进行实验分组:空白对照(Control)组,单纯照射(RT)组,照射+NAC(RT+NAC)组,照射后继续培养24 h后,CCK-8法检测细胞增殖、AnnexinV/PI双标记流式细胞术检测细胞凋亡情况;DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species, ROS)水平以评估细胞内氧化应激程度,比色法测定细胞内谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)含量、超氧化物歧化酶(superoxide dismutase, SOD)活性,Western blot检测Cleaved caspase-3、Bax、Bcl-2蛋白表达变化。结果表明,(1)2 Gy的照射对细胞增殖的影响不明显,当辐射剂量大于2 Gy时,随着辐射剂量的增高,HT22细胞增殖率明显降低(p<0.05);辐射剂量达10 Gy时,细胞增殖抑制率接近50%,因此将10 Gy作为实验最佳辐射剂量。(2)给予10 Gy X射线照射前给予NAC预处理可明显增加HT22细胞的增殖率(p＜0.01)。(3)给予10 Gy X射线照射可明显增加细胞内ROS、MDA含量(p＜0.01),减少细胞内GSH含量和SOD的活力(p＜0.01),促进凋亡蛋白Bax、Cleaved-caspase-3的表达(p＜0.01),细胞凋亡率显著增加(p＜0.01);NAC可减少照射后细胞内ROS和MDA含量(p＜0.01),提高GSH水平及SOD活性(p＜0.01),显著减少凋亡蛋白的表达和细胞凋亡。以上结果表明NAC可抑制辐射相关氧化应激,减少辐射对HT22细胞增殖抑制,减少细胞凋亡。

Effects of N-acetylcysteine on oxidative stress,proliferation and apoptosis of HT22 cells induced by radiation

In order to investigate the effect of N-acetyl-L-cysteine(NAC)on radiation-related oxidative stress and the proliferation and apoptosis of hippocampal neuron HT22 cells,the following process was conducted.First,HT22 cells were irradiated with different doses of X-rays(0,2,4,6,8,10,12 Gy),and the optimum radiation dose(10 Gy)was selected.Then experimental groups were set as follows:control(Control)group,radiation treatment(RT)group,RT+NAC group.Continue to cultivate for 24 h after irradiation,CCK-8 was used to detect cell proliferation;The apoptosis of the HT-22 cell was examined by AnnexinV/PI double labeling flow cytometry.Intracellular reactive oxygen species(ROS)was detected by DCFH-DA fluorescent probe to evaluate intracellular oxidative stress levels;Intracellular glutathione(GSH),malondialdehyde(MDA)content and superoxide dismutase(SOD)activity were detected by colorimetry;The expression of Cleaved caspase-3,Bax and BCl-2 protein was analyzed by Western blot.Results show that:(1)The effect of 2 Gy radiation dose on cell proliferation is not obvious.When the radiation dose was higher than 2 Gy,with the increase of radiation dose the proliferation of HT22 cell was decreased(p<0.05).After radiation dose of 10 Gy,cell proliferation inhibition rate was close to 50%.Therefore,10 Gy was used as the optimal radiation dose for the experiment.(2)Pretreatment with NAC before 10 Gy X-ray irradiation can significantly increase the proliferation rate of HT22 cells.(p<0.01).(3)Radiation treatment can significantly increase intracellular ROS and MDA content(p<0.01),meanwhile intracellular GSH content and SOD activity were reduced(p<0.01).Radiation treatment promoted the expression of apoptotic proteins:Bax and Cleaved caspase-3(p<0.01).The apoptosis rate was increased significantly(p<0.01).NAC can reduce intracellular ROS and MDA content after irradiation(p<0.01).GSH level and SOD activity were increased(p<0.01).The expression of apoptotic proteins and apoptosis rate were reduced significantly.The above results indicate that NAC can inhibit radiation-related oxidative stress,reduce radiation inhibition of HT22 cell proliferation,and reduce apoptosis.