天麻素联合异钩藤碱抑制MPP~+诱导的PC12细胞凋亡的抗氧化机制  

Antioxidant mechanism of gastrodin combined with isorhynchophylline in inhibiting MPP~+-induced apoptosis of PC12 cells

作　　者：李旭 徐天娇[1] 刘立琨[1] 董妙先[1] LI Xu;XU Tian-jiao;LIU Li-kun;DONG Miao-xian(Institute of Medicine,Qiqihar Medical College,Qiqihar 161006,China)

机构地区：[1]齐齐哈尔医学院医药科学研究所,黑龙江齐齐哈尔161006

出　　处：《中国中药杂志》2021年第2期420-425,共6页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81873133)。

摘　　要：天麻-钩藤药对是临床治疗帕金森病出现频次最高的药对,天麻素和异钩藤碱是天麻-钩藤药对抗帕金森病的主要有效成分。为了观察天麻素联合异钩藤碱对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP^(+))诱导的PC12细胞凋亡的影响及探讨其抗氧化机制,该研究采用分光光度法检测细胞上清液乳酸脱氢酶(lactate dehydrogenase,LDH)含量,流式细胞术检测细胞凋亡,碘化丙啶(propidium iodide,PI)单染法检测细胞周期分布,分光光度法检测脂质过氧化物(lipid peroxidation,LPO)含量,实时定量PCR法检测细胞caspase-3 mRNA表达,免疫印迹法检测血红素加氧酶-1(heme oxygenase 1,HO-1)和醌氧化还原酶-1(NADPH:quinone oxidore-ductase 1,NQO-1)蛋白表达。结果发现,天麻素联合异钩藤碱显著降低MPP^(+)诱导的PC12细胞FITC-Annexin V阳性细胞比率和细胞周期阻滞,显著下调MPP^(+)诱导的PC12细胞caspase-3 mRNA表达,显著降低MPP^(+)诱导的PC12细胞LPO含量,上调HO-1和NQO-1蛋白表达。值得注意的是,天麻素联合异钩藤碱的这些药理作用可被细胞外信号调节激酶1/2(extracellular signal-regulated kinases 1/2,ERK1/2)抑制剂PD98059、磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)抑制剂LY294002或/和糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)抑制剂氯化锂预处理逆转,提示天麻素联合异钩藤碱通过ERK1/2和PI3K/GSK-3β信号通路抑制MPP^(+)诱导的PC12细胞凋亡和氧化应激。结果表明,天麻素联合异钩藤碱上调MPP^(+)诱导的PC12细胞HO-1和NQO-1蛋白表达,进而减少氧化应激,抑制其凋亡。Gastrodiae Rhizoma-Uncariae Ramulus cum Uncis is the most frequently used herbal pair in the treatment of Parkinson′s disease(PD).Gastrodin and isorhynchophylline are important components of Gastrodiae Rhizoma-Uncariae Ramulus cum Uncis herb pair with anti-Parkinson mechanism.This study aimed to investigate the effect of gastrodin combined with isorhynchophylline on 1-methyl-4-phenylpyridinium(MPP^(+))-induced apoptosis of PC12 cells and their antioxidant mechanism.The leakage of lactate dehydrogenase(LDH)from cells to media was analyzed by spectrophotometry.Apoptotic cells were labeled with Annexin V-fluorescein isothiocyanate(FITC)and propidium iodide(PI)and analyzed by flow cytometry.The cell cycle was analyzed using propidium iodide(PI)staining.Lipid peroxidation(LPO)level was analyzed by spectrophotometry.The mRNA expression of caspase-3 was examined by Real-time RT-PCR.The protein expressions of heme oxygenase 1(HO-1)and NADPH:quinoneoxidore-ductase 1(NQO-1)were determined by Western blot.Gastrodin combined with isorhynchophylline reduced the percentage of Annexin V-positive cells and cell cycle arrest in MPP^(+)-induced PC12 cells.Gastrodin combined with isorhynchophylline down-regulated the mRNA expression of caspase-3,up-regulated the protein expressions of HO-1 and NQO-1,and reduced LPO content in MPP^(+)-induced PC12 cells.PD98059,LY294002 or LiCl could partially reverse these changes pretreated with gastrodin combined with isorhynchophylline,suggesting that gastrodin combined with isorhynchophylline inhibited MPP^(+)-induced apoptosis of PC12 cells and oxidative stress through ERK1/2 and PI3 K/GSK-3βsignal pathways.Our experiments showed that gastrodin combined with isorhynchophylline could down-re-gulate the mRNA expression of caspase-3 and up-regulate the protein expressions of HO-1 and NQO-1,so as to reduce oxidative stress and inhibit apoptosis.

关 键 词：天麻素 异钩藤碱 帕金森病 凋亡 氧化应激 

分 类 号：R285.5[医药卫生—中药学]