艾烟可吸入物对大鼠神经细胞氧化损伤的影响
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摘要
目的探讨艾烟可吸入物(PM10)对大鼠神经细胞氧化损伤的影响及PI3K/Akt信号通路在其中的作用。方法采用24 h内新生SD大鼠乳鼠前额叶皮质及海马组织体外培养原代神经细胞,H_2O_2诱导建立氧化损伤细胞模型;随机分为空白组、氧化损伤组、氧化损伤+PM10组、氧化损伤+PM10+抑制剂组、PM10组。采用TUNEL和DAPI染色观察神经细胞凋亡形态;MTT法及Western blot分别检测细胞存活率及目的蛋白表达,并用PI3K/Akt通路抑制剂LY294002验证作用通路。结果 MTT检测显示,200μmol/L H_2O_2能降低神经细胞50%存活率(P<0.001),0.1、0.5 ng/mL艾烟PM10能明显抑制H_2O_2诱导氧化损伤的神经细胞凋亡(P<0.001);TUNEL和DAPI染色显示,0.5μg/m L艾烟PM10能拮抗H_2O_2诱导氧化损伤神经细胞凋亡的形态学变化(P<0.001);Western blot检测显示,H_2O_2及LY294002能降低p-Akt、Bcl-2的表达(P<0.05,P<0.001),上调active-Caspase-3的表达(P<0.01,P<0.001),而艾烟PM10作用相反(P<0.01,P<0.001)。结论艾烟PM10能减少神经细胞氧化应激的凋亡,其机制可能是激活PI3K/Akt信号通路并调控下游凋亡蛋白Bcl-2、active-Caspase-3的表达,拮抗细胞凋亡。
Objective To investigate the effects of inhalation of Moxa smoke on oxidative damage of neuronsand the role of PI3K/Akt signaling pathway. Methods Primary cultured neurons were cultured in the prefrontal cortex and hippocampus of neonatal SD rats within 24 hours. H_2O_2 was used to establish the oxidative damage cell model. They were randomly divided into blank group, injury group, PM10 + injury group, PM10 + injury + inhibitor group and PM10 group. Morphological changes of neuronal apoptosis were observed by TUNEL and DAPI staining. Cell viability and protein expression were detected by MTT and Western blot, and the pathway was verified by PI3K/Akt pathway inhibitor LY294002. Results MTT showed that 200 μmol/L H_2O_2 could reduce the 50% survival rate of neurons(P<0.001). 0.1, 0.5 ng/m L Moxa smoke PM10 could significantly inhibit the apoptosis of neurons induced by H_2O_2(P<0.001). TUNEL and DAPI staining showed that 0.5 μg/m L Moxa smoke PM10 could antagonize the morphological changes of H_2O_2-induced oxidative damage in neuronal apoptosis(P<0.001); Western blot showed that H_2O_2 and LY294002 could decrease the expressionsof p-Akt and Bcl-2(P<0.05, P<0.001), and up-regulated the expression of active-Caspase-3(P<0.01, P<0.001), while the effect of PM10 was opposite(P<0.01, P<0.001). Conclusion Moxa smoke PM10 can reduce the apoptosis of neurons under oxidative stress by activating PI3K/Akt signaling pathway and regulating the expressions of downstream apoptotic proteins Bcl-2 and active-Caspase-3.
Objective To investigate the effects of inhalation of Moxa smoke on oxidative damage of neuronsand the role of PI3K/Akt signaling pathway. Methods Primary cultured neurons were cultured in the prefrontal cortex and hippocampus of neonatal SD rats within 24 hours. H_2O_2 was used to establish the oxidative damage cell model. They were randomly divided into blank group, injury group, PM10 + injury group, PM10 + injury + inhibitor group and PM10 group. Morphological changes of neuronal apoptosis were observed by TUNEL and DAPI staining. Cell viability and protein expression were detected by MTT and Western blot, and the pathway was verified by PI3K/Akt pathway inhibitor LY294002. Results MTT showed that 200 μmol/L H_2O_2 could reduce the 50% survival rate of neurons(P<0.001). 0.1, 0.5 ng/m L Moxa smoke PM10 could significantly inhibit the apoptosis of neurons induced by H_2O_2(P<0.001). TUNEL and DAPI staining showed that 0.5 μg/m L Moxa smoke PM10 could antagonize the morphological changes of H_2O_2-induced oxidative damage in neuronal apoptosis(P<0.001); Western blot showed that H_2O_2 and LY294002 could decrease the expressionsof p-Akt and Bcl-2(P<0.05, P<0.001), and up-regulated the expression of active-Caspase-3(P<0.01, P<0.001), while the effect of PM10 was opposite(P<0.01, P<0.001). Conclusion Moxa smoke PM10 can reduce the apoptosis of neurons under oxidative stress by activating PI3K/Akt signaling pathway and regulating the expressions of downstream apoptotic proteins Bcl-2 and active-Caspase-3.
引文
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[15]黄畅,崔莹雪,刘钧天,等.艾灸及艾烟对载脂蛋白E基因敲除小鼠血清MDA、SOD水平的影响[J].世界中医药,2016,11(8):1407-1409,1413.
[2]YU M,JIN R,ZHAO B,et al.SPME-GC-MS determination of phenol in the smoke of moxa[J].Chinese Journal of Pharmaceutical Analysis,2012,32(10):1870-1873.
[3]XU H,ZHAO B,CUI Y,et al.Effects of moxa smoke on monoamine neurotransmitters in SAMP8 mice[J].Evid Based Complement Alternat Med,2013,2013:178067.
[4]HUANG J,LIN M Y,ZHAO B,et al.PM10 mass concentration and oxidative capacity of moxa smoke.[J].Qjm,2015,108(9):705-710.
[5]HITOSUGI N,OHNO R,HATSUKARI I,et al.Diverse biological activity of Moxa extract and smoke[J].Vivo,2001,15(3):249-254.
[6]SAKAGAMI H,MATSUMOTO H,SATOH K,et al.Cytotoxicity and radical modulating activity of Moxa smoke[J].Vivo,2005,19(2):391-397.
[7]MATSUMOTO H,SHIMADA J,NAGASAKA H,et al.Inhibition by Moxa smoke of NO production and iNOS expression in mouse macrophagelike cells Raw 264.7[J].Vivo,2005,19(2):471.
[8]MENG X N,HUAN-FANG X U,CUI Y X.Effect of moxa products after burning on SOD,MDA and GSH-Px in the brain of senescence accelerated mice[J].Global Traditional Chinese Medicine,2011,4(6):413-415.
[9]刘耀萌,刘钧天,黄畅,等.不同艾灸因素对阿尔茨海默小鼠PI3K/AKT通路与皮质β淀粉样蛋白沉淀的影响[J].世界中医药,2016,11(8):1395-1400.
[10]苏倩.Akt信号通路通过激活TFEB而抑制氧化应激诱导的神经细胞凋亡[D].石家庄:河北医科大学,2017.
[11]朱若岑,蒋维维,谭柱良,等.动物体内活性氧、氧化应激与细胞凋亡以及抗氧化剂研究进展[J].中兽医医药杂志,2015,34(3):21-25.
[12]塔依尔·吐尔松,买吾拉尼江·依孜布拉,努尔买买提·艾买提.神经退行性疾病的抗氧化研究进展[J].新疆医科大学学报,2017,40(6):725-729.
[13]刘钧天,黄畅,刘耀萌,等.艾灸各因素对APP/PS-1双转基因AD小鼠脑中DNA、蛋白质、脂质氧化应激的影响[J].中华中医药杂志,2015,40(5):1375-1379.
[14]蔡虹,邬继红,赵百孝,等.艾烟冷凝物对大鼠肺泡巨噬细胞NR8383活性和吞噬功能的影响[J].北京中医药大学学报,2013,36(7):501-504.
[15]黄畅,崔莹雪,刘钧天,等.艾灸及艾烟对载脂蛋白E基因敲除小鼠血清MDA、SOD水平的影响[J].世界中医药,2016,11(8):1407-1409,1413.