基于MAPK信号通路研究艾柱燃烧产物减轻蓝光诱导ARPE-19细胞损伤的作用机制

Mechanism of moxa smoke extract on attenuating blue light induced ARPE-19 cell injury based on MAPK signaling pathway

目的探讨艾柱燃烧产物(moxa smoke extract,MSE)对蓝光诱导人视网膜色素上皮(adult retinal pigment epithelium-19,ARPE-19)细胞损伤的保护作用及其作用机制。方法体外培养ARPE-19细胞,CCK-8法检测不同质量浓度(0.25、0.50、1.00、2.00、4.00、5.00、10.00、50.00、100.00、150.00μg/mL)MSE对ARPE-19细胞活力的影响。设置对照组、对照+MSE高剂量组、蓝光组、蓝光+叶黄素组、蓝光+MSE低剂量组和蓝光+MSE高剂量组。对照组和蓝光组加入空白培养基,其余4组分别加入相应的含药培养基干预24 h后,除对照组以及对照+MSE高剂量组外,各组均在蓝光(430 nm,96.1 W/m2)下刺激1 h。建立ARPE-19细胞损伤模型后,采用乳酸脱氢酶(lactate dehydrogenase,LDH)法检测质膜完整性;采用倒置荧光显微镜观察细胞形态学变化;采用流式细胞仪检测细胞内活性氧(reactive oxygen species,ROS)水平;采用试剂盒检测细胞内氧化应激指标丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)水平和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、过氧化氢酶(catalase,CAT)以及超氧化物歧化酶(superoxide dismutase,SOD)活性;采用Western blotting法检测丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路相关蛋白表达。结果与对照组相比,MSE(0.25~4.00μg/mL)对ARPE-19细胞活力没有显著影响。与蓝光组相比,MSE可以显著降低培养上清中的LDH活性(P<0.01),减轻质膜损伤以及细胞形态学变化,调节MAPK信号通路关键蛋白Ras、磷酸化c-Raf(phosphorylated c-Raf,p-c-Raf)、磷酸化细胞外调节蛋白激酶1/2(phosphorylated extracellular regulated protein kinase 1/2,p-Erk1/2)、磷酸化丝裂原活化的细胞外信号调节激酶1/2(phosphorylated mitogen-activated extracellular signal-regulated kinase 1/2,p-MEK1/2)和p-p38 MAPK表达(P<0.05、0.01),调节细胞内SOD、CAT、GSH-Px活性以及GSH、MDA、ROS水平(P<0.05、0.01),改善ARPE-19细胞氧化应激反应。结论MSE可能通过调控ROS介导的MAPK信号通路相关蛋白的表达,对蓝光诱导的ARPE-19细胞氧化应激损伤起保护作用。

Objective To investigate the effect and mechanism of moxa smoke extract(MSE)on blue light-induced ARPE-19 cells injury.Methods ARPE-19 cells were cultured in vitro,and effect of MSE at different concentrations(0.25,0.50,1.00,2.00,4.00,5.00,10.00,50.00,100.00,150.00μg/mL)on viability of ARPE-19 cells was detected by CCK-8 method.Control group,control+MSE high-dose group,blue-light group,blue-light+lutein group,blue-light+MSE low-dose group and blue-light+MSE high-dose group were set.Blank medium was added to control group and blue light group,and corresponding drug-containing medium was added to the other four groups,after intervention for 24 h,except for control group and control+MSE high-dose group,all groups were stimulated under blue light(430 nm,96.1 W/m2)for 1 h.After ARPE-19 cell injury model was established,integrity of plasma membrane was detected by lactate dehydrogenase(LDH)method;Morphological changes of cells were observed by inverted fluorescence microscope;Intracellular reactive oxygen species(ROS)level were detected by flow cytometry species;Kits were used to detect intracellular oxidative stress indicators malondialdehyde(MDA),glutathione(GSH)levels and glutathione peroxidase(GSHPx),catalase(CAT)and superoxide dismutase(SOD)activities;Western blotting was used to detect mitogen-activated protein kinases(MAPK)signaling pathway-related proteins expressions.Results Compared with control group,MSE(0.25—4.00μg/mL)had no significant effect on viability of ARPE-19 cells.Compared with blue light group,MSE significantly reduced LDH activity in culture supernatant(P<0.01),alleviated the damage of plasma membrane and cell morphological changes,regulated key proteins expressions of MAPK signaling pathway such as Ras,phosphorylated c-Raf(p-c-Raf),phosphorylated extracellular regulated protein kinase 1/2(p-Erk1/2),phosphorylated mitogen-activated extracellular signal-regulated kinase 1/2(p-MEK1/2)and p-p38 MAPK(P<0.05,0.01),regulated intracellular SOD,CAT,GSH-Px activities and GSH,MDA,ROS levels(P<0.05,0.01),and improved the oxidative stress response of ARPE-19 cells.Conclusion MSE may protect ARPE-19 cells from blue light-induced oxidative stress injury by regulating the expression of ROS-mediated MAPK signaling pathway-related proteins.