摘要
目的探讨槲皮素对H_(2)O_(2)损伤的大鼠嗜铬细胞瘤(PC12)细胞的保护作用及机制。方法先选取不同浓度H_(2)O_(2)(100、200、300、400、500μmol/L)干预PC12细胞,确定氧化损伤模型的条件,然后将PC12细胞分为对照组、模型组和槲皮素(6.25、12.5、25μmol/L)组。通过MTS法、Hoechst染色法检测细胞存活和凋亡;镜下观察细胞形态;免疫荧光检测8-羟脱氧鸟苷(8-OHdG);ELISA法检测丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH);Western blot法检测B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、蛋白激酶B(Akt)和磷酸化蛋白激酶B(p-Akt)表达水平。结果与对照组相比,模型组用500μmol/L H_(2)O_(2)干预6 h后,细胞存活率下降至50%(P<0.01),凋亡率上升为38%(P<0.01);镜下可见细胞萎缩变圆;8-OHdG表达增加(P<0.05);GSH活性降低,MDA含量升高(P<0.05);Bcl-2、Akt、p-Akt表达减少,Bax表达增加(P<0.05)。与模型组相比,槲皮素各浓度组细胞存活率均上升(P<0.05),凋亡率下降(P<0.01);细胞突触再现;8-OHdG表达降低(P<0.05);GSH活性升高,MDA含量下降(P<0.05);Bcl-2、Akt、p-Akt蛋白上调,Bax蛋白下调(P<0.05)。结论槲皮素能拮抗H_(2)O_(2)诱导的PC12细胞氧化损伤,其作用机制可能与Akt调控的通路以及抗氧化酶的相关作用有关。
Objective To investigate the protective effect and its mechanisms of quercetin in rat pheochromocytoma(PC12cell)cell injury stimulated by H_(2)O_(2).Methods Different concentrations of H_(2)O_(2)(100,200,300,400,500μmol/L)were used to interfere with PC12 cells to construct oxidative stress model,then the experiments were divided into control group,model group and quercetin group(6.25,12.5,25μmol/L).The cell survival and apoptosis were detected by MTS and Hoechst,respectively.Morphology was observed by microscope,and 8-hydroxydeoxyguanosine(8-OHdG)expression was measured by immunofluorescence.Furthermore,the levels of malondialdehyde(MDA)and glutathione(GSH)were assayed by ELISA and the expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),protein kinase B(Akt)and phosphorylated protein kinase B(p-Akt)were evaluated by Western blot.Results After constructed via 500μmol/L H_(2)O_(2)for 6 h,compared with the control group,cell survival rate in model group was decreased to 50%(P<0.01),apoptosis rate was increased to 38%(P<0.01);microscopically,the cells became round and atrophic;8-OHdG expression increased(P<0.05);GSH activity was reduced,and the content of MDA was increased(P<0.05);Bcl-2,Akt and p-Akt expression levels were decreased,and Bax expression was increased(P<0.05).Compared with the model group,the cell survival rate of quercetin groups increased(P<0.05)and apoptosis rate decreased(P<0.01);cell synapse reappeared;the expression of 8-OHdG decreased(P<0.05);the level of GSH increased and the level of MDA decreased(P<0.05);the expression levels of Bcl-2,Akt and p-Akt were up-regulated,and the expression of Bax was down-regulated(P<0.05).Conclusion Quercetin could relieve oxidative damage of PC12 cells induced by H_(2)O_(2).Its mechanism may be related to Akt regulated pathway and the related effects of antioxidant enzymes.
作者
易鹏吉
张哲宇
彭伟军
YI Pengji;ZHANG Zheyu;PENG Weijun(Department of Integrated Traditional Chinese and Western Medicine,The Second Xiangya Hospital,Central South University,Changsha,Hunan 410011,China)
出处
《湖南中医药大学学报》
CAS
2021年第6期821-826,共6页
Journal of Hunan University of Chinese Medicine
基金
国家自然科学基金面上项目(81873169)
湖南省自然科学基金面上项目(2020JJ4803)
湖南省中医药科研计划项目(201225)。
