氧化应激在鱼藤素致SH-SY5Y细胞神经毒性中的作用

Effect of oxidative stress in SH-SY5Y cell injury induced by deguelin

目的研究氧化应激与鱼藤素致神经毒性的相关性,为后续鱼藤素的结构改造和联合用药提供机制基础。方法将鱼藤素(1.56~100μmol·L-1)与SH-SY5Y细胞共孵育培养24~72 h,采用CCK-8法测定细胞存活率;比色法测定乳酸脱氢酶(lactate dehydrogenase,LDH)漏出量、丙二醛(malondialdehyde,MDA)含量、谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)和超氧化物歧化酶(superoxide dismutase,SOD)的活力;流式细胞术检测活性氧(reactive oxygen species,ROS)含量;免疫印迹法检测细胞中MEK、EGF、RAS、CREB蛋白的表达情况。结果鱼藤素对SH-SY5Y细胞增殖有抑制作用,且呈浓度和时间依赖性(P<0.05)。鱼藤素损伤细胞后,LDH漏出量、MDA和ROS含量明显增加,GPx和SOD的活力下降(P<0.05)。同时MAPK/ERK信号通路中MEK、EGF、RAS、CREB蛋白水平出现不同程度的下调。结论鱼藤素致SH-SY5Y神经细胞的损伤与氧化应激密切相关,MAPK/ERK信号通路可能在其中起介导作用。

Aim To study the correlation between oxidative stress and neurotoxicity induced by deguelin,providing the mechanism basis for the structural modification and drug combination about deguelin.Methods SH-SY5Y cells were exposed to deguelin ranging from 1.56~100μmol·L-1 for 24 to 72 h,whose survival rate was determined by CCK-8 assay.The content of LDH,MDA levels,GPx and antioidant enzyme activities of SOD were determined using the assay kits according the manufacturer’s protocol.And the content of ROS was determined by flow cytometry.The expressions of MEK,EGF,ERK and CREB were determined by immunoblotting.Results Deguelin obviously inhibited the growth of SH-SY5Y cells in a concentration-and time-dependent manner(P<0.05).After the cells were injured by deguelin,the content of ROS,LDH and MDA in SH-SY5Y cells increased obviously(P<0.05),while the vitality of GPx and SOD decreased obviously(P<0.05)in a dose-dependent manner.Furthermore,the protein expression levels of MEK,EGF,RAS and CREB in MAPK/ERK signaling pathway decreased obviously when treated with deguelin in a dose-dependent manner.(P<0.05).Conclusions The trauma of SH-SY5Y cells induced by deguelin is closely related to oxidative stress,in which MAPK/ERK signaling pathway may play a mediating role.