亚甲基蓝类似物抗谷氨酸诱导HT22细胞氧化损伤及其机制  

Effect of methylene blue analogues on glutamate-induced HT22 neuron cell injury and its mechanism

作　　者：陈紫微 陈秋荷 蔡瑜 侯姗姗 刘福和 CHEN Ziwei;CHEN Qiuhe;CAI Yu;HOU Shanshan;LIU Fuhe(Pharmaceutical Department,Zhejiang pharmaceutical college,Ningbo 315000,China;School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,China)

机构地区：[1]浙江药科职业大学药学院,浙江宁波315000 [2]广州中医药大学中药学院,广东广州510006

出　　处：《温州医科大学学报》2022年第12期956-964,971,共10页Journal of Wenzhou Medical University

基　　金：浙江省基础公益研究计划项目(LGF18H090015,LGF21H090016)。

摘　　要：目的:探讨亚甲基蓝(MB)类似物[2-氯吩噻嗪(2-C)、天青A(A-A)、天青B(A-B)、中性红(NR)]抗谷氨酸(Glu)诱导HT22细胞氧化损伤及其作用机制。方法:采用MTT法检测MB、2-C、A-A、A-B、NR在HT22细胞中的毒性;采用MTT法、LDH法检测各个化合物对Glu诱导HT22细胞氧化损伤模型的保护作用;采用H_(2)DCF-DA染色剂检测各组细胞中ROS水平;采用siRNA干扰技术,抑制HT22细胞内MEF2D表达;采用Western blot法检测各个化合物对HT22细胞中肌细胞增强因子2D(MEF2D)、NADH脱氢酶6(ND6)、Akt、GSK-3β蛋白变化情况。结果:根据MTT实验结果计算各个化合物LD_(50):MB(50.66μmol/L)、2-C(175.90μmol/L)、A-A(152.40μmol/L)、A-B(140.40μmol/L)、NR(73.19μmol/L)。除NR外,其余化合物均对Glu诱导的HT22细胞氧化损伤具有显著的细胞保护作用,随后计算各个化合物EC_(50)值得出:MB(42.13 nmol/L)、2-C(34.32 nmol/L)、A-A(48.96 nmol/L)、A-B(44.42 nmol/L);ROS染色结果显示:100 nmol/L的MB、2-C、A-B和A-A能显著降低Glu诱导的HT22细胞中ROS水平,而NR对Glu诱导的HT22细胞中ROS水平上升无明显作用;Western blot检测结果证明MB、A-B和A-A明显提高HT22细胞内MEF2D的蛋白水平(P<0.05);采用siRNA干扰技术抑制HT22细胞内MEF2D表达后,MTT结果显示抑制MEF2D表达能部分取消化合物MB、2-C、A-B和A-A对Glu诱导HT22细胞损伤的保护作用(P<0.05);MB、2-C、A-B和A-A能够激活Akt,增加GSK-3β磷酸化(P<0.05),使GSK-3β失活,这可能与其激活MEF2D进而提高ND6活性有关。结论:2-C与MB相比具有更低的细胞毒性,且其抗Glu诱导细胞损伤作用效价强于MB;具有吩噻嗪结构的化合物:MB、2-C、A-B和A-A可能通过调节Akt/GSK-3β通路,提高MEF2D/ND6活性,改善线粒体呼吸链功能,进而产生抗Glu诱导的神经细胞氧化损伤作用。Objective:To investigate the effect of methylene blue(MB)analogues(2-Chlorophenothiazine,Neutral red,Azure A and Azure B)on glutamate-induced HT22 neuronal cell injury and its mechanism.Methods:The toxicity effect of 2-Chlorophenothiazine(2-C),Azure A(A-A),Azure B(A-B)and Neutral red(NR)on HT22 neuronal cells were detected by MTT.The protect effect of methylene blue analogues against glutamateinduced cell death was detected by MTT and LDH release assay;ROS production was measured by H_(2)DCF-DA staining;siRNA was used to download the expression of MEF2D.Western blot was used to detect the protein levels of MEF2D,ND6,Akt and GSK-3β.Results:LD_(50) of compound was calculated according to the results of MTT experiment,:MB(50.66μmol/L),2-C(175.90μmol/L),A-A(152.40μmol/L),A-B(140.40μmol/L),NR(73.19μmol/L).Except for the NR,the other compounds had significant protective effect on glutamate-induced oxidative damage of HT22 cells.The EC_(50) value of each compound was calculated as follows:MB(42.13 nmol/L),2-C(34.32 nmol/L),A-A(48.96 nmol/L),A-B(44.42 nmol/L).MB,2-C,A-B and A-A significantly reduced glutamate-induced ROS generation in HT22 cells detected by H_(2)DCF-DA staining at 100 nmol/L.However,NR had no obvious effect on glutamate-induced ROS generation in HT22 cells.MB,2-C,A-B and A-A elevated the protein level of MEF2D(P<0.05).To investigate the role of MEF2D in the neuroprotective effect of MB analogues against glutamate-induced cell death,we knocked down MEF2D by siRNA in HT22 cells(P<0.05).MTT results showed that the knockdown of MEF2D abolished part of neuroprotective effect of MB,2-C,A-B and A-A against glutamate in HT22 cells.Further studies on the mechanism showed that 2-C,A-B,A-A and MB could activate Akt,increase GSK-3βphosphorylation(P<0.05)and inactivate GSK-3β,which may be related to the activation of MEF2D and ND6.Conclusion:Compared with MB,2-C has lower cytotoxicity,and stronger damage activity to the anti-glutamate-induced cells.Compounds with phenothiazine structure such as 2-C,A-B,A-A an

关 键 词：亚甲基蓝类似物 MEF2D 氧化损伤 神经退行性疾病 

分 类 号：R96[医药卫生—药理学]