姜酮通过激活Nrf2/HO-1信号通路减轻OGD/R后氧化应激损伤对HT22细胞凋亡的抑制作用

Inhibitory effect of gingerone on apoptosis of HT22 cells by alleviation oxidative stress damage after OGD/R through activating Nrf2/HO-1 signaling pathway

摘要

目的 探讨姜酮对氧糖剥夺/复糖复氧（OGD/R）后小鼠海马神经元HT22细胞的保护作用，阐明其相关作用机制。 方法 培养HT22细胞，设置不同OGD/R时间梯度，建立OGD/R细胞损伤模型。HT22细胞分为对照组、OGD/R组、OGD/R＋1 μmol·L^－1姜酮组、OGD/R＋10 μmol·L^－1姜酮、OGD/R＋100 μmol·L^－1姜酮组和OGD/R+0.2%二甲亚枫（DMSO）组，CCK-8法检测各组细胞活性并计算各组细胞存活率，确定姜酮最适药物浓度。细胞分为对照组、OGD/R组、OGD/R+姜酮组和OGD/R+姜酮+核因子E2相关因子2（Nrf2）抑制剂（ML385）组，OGD/R+姜酮组细胞经姜酮给药处理4 h后予以OGD 8 h和复糖复氧8 h 处理，OGD/R+姜酮+ML385组细胞在姜酮给药前予以10 μmol·L^－1 ML385预处理6 h，CCK-8法检测各组细胞活性，Western blotting法检测各组细胞中Nrf2、血红素加氧酶1（HO-1）、B细胞淋巴瘤2（Bcl-2）和Bcl-2相关X蛋白（Bax）蛋白表达水平，酶联免疫吸附试验（ELISA）法检测各组细胞培养上清中超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平。 结果 与对照组比较，HT22细胞经OGD 8 h和复糖复糖8 h处理后细胞存活率低于50%，以OGD 8 h和复糖复糖8 h建立HT22细胞OGD/R模型。与OGD/R组比较，OGD/R+不同剂量姜酮组细胞存活率均不同程度升高，其中OGD/R+100 μmol·L^－1姜酮组细胞存活率升高最明显（P<0.01），故选用100 μmol·L^－1姜酮用于后续实验。与对照组比较，OGD/R组细胞活性明显降低（P<0.01），细胞中Nrf2、HO-1和Bax蛋白表达水平明显升高（P<0.01），Bcl-2蛋白表达水平明显降低（P<0.05），细胞培养上清中SOD活性明显降低（P<0.01），MDA水平明显升高（P<0.01）；与OGD/R组比较，OGD/R+姜酮组细胞活性明显升高（P<0.01），细胞中Nrf2、HO-1和Bcl-2蛋白表达水平明显升高（P<0.05或P<0.01），Bax蛋白表达水平明显降低（P<0.05），细胞培养上清中SOD活性明显升高（P<0.01），MDA水平明显降低（P<0.01）；与OGD/R+姜酮组比较，OGD/R+姜酮+ML385组细胞活性明显降低（P<0.01），细胞中Nrf2、HO-1和Bcl-2蛋白表达水平明显降低（P<0.01），Bax蛋白表达水平明显升高（P<0.01），细胞培养上清中SOD活性明显降低（P<0.01），MDA水平明显升高（P<0.05）。 结论 姜酮可通过激活Nrf2/HO-1信号通路减轻OGD/R后氧化应激损伤对HT22细胞凋亡的抑制作用。

Abstract

Objective To discuss the protective effect of gingerone on the hippocampal neuron HT22 cells after oxygen-glucose deprivation/reoxygenation （OGD/R），and to clarify the related mechanism. Methods The HT22 cells were cultured， and the OGD/R cell injury model was established by setting the gradient of OGD/R time. The HT22 cells were divided into control group，OGD/R group， OGD/R+1 μmol·L^－1 gingerone group， OGD/R + 10 μmol·L^－1 gingerone group， OGD/R+100 μmol·L^－1 gingerone group，and OGD/R+0.2% dimethyl sulfoxide（DMSO） group.The viability of the cells in various groups was detected by CCK-8 assay； the survival rates of the cells in various groups were calculated to determine the optimal drug concentration of gingerone. The cells were divided into control， OGD/R group， OGD/R+ gingerone， and OGD/R+gingerone+nuclear factor erythroid-2-related factor 2（Nrf2） inhibitor（ML385） groups.The cells in OGD/R + gingerone group were treated with gingerone for 4 h before OGD treatment for 8 h followed by reoxygenation for 8 h， and the cells in OGD/R+gingerone+ML385 group were treated with 10 μmol·L^－1 ML385 for 6 h before gingerone treatment. The viability of the cells in various groups was detected by CCK-8 assay；the expression levels of Nrf2， heme oxygenase-1 （HO-1）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） proteins in the cells in various groups were detected by Western blotting method；the activity of superoxide dismutase （SOD） and the level of malondialdehyde （MDA） in the cell culture supernatant in various groups were detected by enzyme-linked immunosorbent assay （ELISA） method. Results Compared with control group， the survival rate of the HT22 cells was below 50% after treated with OGD for 8 h and reoxygenation for 8 h， so the HT22 cell OGD/R model was established by treated with OGD for 8 h and reoxygenation for 8 h. Compared with OGD/R group，the survival rates of the cells in OGD/R+different doses of gingerone groups were increased to various extents， and the survival rate of the cells in OGD/R+ 100 μmol·L^－1 gingerone group was significantly increased （P<0.01）； so 100 μmol·L^－1 gingerone was used for the subsequent experiment. Compared with control group， the viability of the cells in OGD/R group was significantly decreased （P<0.01）， and the expression levels of Nrf2， HO-1， and Bax proteins in the cells were significantly increased （P<0.01）， while the expression level of Bcl-2 protein in the cells was significantly decreased （P<0.05）， and the SOD activity in the cell culture supernatant was significantly decreased （P<0.01）， and the level of MDA was significantly increased （P<0.01）； compared with OGD/R group， the viability of the cells in OGD/R + gingerone group was significantly increased （P<0.01）， and the expression levels of Nrf2， HO-1， and Bcl-2 proteins in the cells were significantly increased （P<0.05 or P<0.01）， while the expression level of Bax protein in the cells was decreased（P<0.05）， the SOD activity in the cell culture supernatant was significantly increased （P<0.01）， and the level of MDA was significantly decreased （P<0.01）； compared with OGD/R + gingerone group， the viability of the cells in OGD/R + gingerone + ML385 group was significantly decreased （P<0.01）， and the expression levels of Nrf2， HO-1， and Bcl-2 proteins were significantly decreased （P<0.01）， while the expression level of Bax protein in the cells was significantly increased （P<0.01），the SOD activity in the cell culture supernatant was significantly decreased （P<0.01）， and the level of MDA was significantly increased （P<0.05）. Conclusion Gingerone alleviates the oxidative stress damage，and thereby plays an inhibiory effect on the apoptosis of the HT22 neurons by activating the Nrf2/HO-1 signaling pathway after OGD/R.

关键词

姜酮 / 糖氧剥夺 / HT22神经元 / 核因子E2相关因子2 / 血红素加氧酶1 / 氧化应激 / 细胞凋亡

Key words

Gingerone / Oxygen-glucose deprivation / HT22 neuron / Nuclear factor erythroid-2-related factor 2 / Heme oxygenase 1 / Oxidative stress / Apoptosis

Author summay

侯玮琛（1987－），女，河北省石家庄市人，主治医师，医学博士，主要从事神经内科基础和临床方面的研究。