Sulforaphane抑制热灭活大肠杆菌诱导的HeLa细胞凋亡  

Sulforaphane inhibits apoptosis of heat-killed E.coli-induced HeLa cell

作　　者：张箭 董艳艳[2] 瞿丛 王瑶 赵朔暄[2] 赵文然 Zhang Jian;Dong Yanyan;Qu Cong;Wang Yao;Zhao Shuoxuan;Zhao Wenran(Function Experiment Teaching Center of Harbin Medical University,Harbin 150081 China;Department of Cell Biology,Harbin Medical University,Harbin 150081,China)

机构地区：[1]哈尔滨医科机能实验教学中心,150081 [2]哈尔滨医科大学细胞生物学教研室,150081

出　　处：《国际免疫学杂志》2019年第3期235-240,共6页International Journal of Immunology

基　　金：国家自然科学基金(81672007).

摘　　要：目的研究Sulforaphane(SFN)对细胞凋亡的抑制作用及其在脓毒症中对细胞的保护作用。方法将HeLa细胞分为4组:对照组、热灭活大肠杆菌(Escherichia coli,E.coli)处理组、SFN处理组(SFN组)和热灭活E.coli+SFN组。酶联免疫反应检测炎症因子白细胞介素(interleukin,IL)-1β和肿瘤坏死因子(tumor necrosis factor,TNF)-α;流式细胞术(annexin V/propidium iodide双染)检测细胞凋亡;免疫荧光检测细胞内活性氧(reactive oxygen species,ROS)含量及线粒体膜电位的变化;Western blot检测活化的caspase 3、bax和bcl-2水平。结果热灭活E.coli处理后,细胞产生的炎症因子IL-1β[(121.13±8.44)pg/mL比(36.67±5.82)pg/mL,P<0.05]和TNF-α[(123.83±7.32)pg/mL比(20.63±1.91)pg/mL,P<0.05]明显升高、凋亡细胞数明显增加[(32.49±3.96)%比(12.52±1.37)%,P<0.05]、活化的caspase 3[(2.28±0.19)比(1.01±0.19),P<0.05)]和BAX[(4.81±0.80)比(1.04±0.25),P<0.05)]水平明显升高,而bcl-2[(0.27±0.08)比(0.96±0.18),P<0.05)]水平明显降低。用SFN处理后(包括SFN组、热灭活E.coli+SFN组),IL-1β[(45.41±13.16)pg/mL比(121.13±8.44)pg/mL,P<0.05],TNF-α[(30.3±6.0)pg/mL比(123.8±7.3)pg/mL,P<0.05]水平明显降低、凋亡细胞明显减少[(11.34±2.05)%比(32.49±3.96)%,P<0.05]、活化的caspase3[(1.01±0.07)比(2.282±0.19),P<0.05)]、BAX[(2.15±0.14)比(4.81±0.80),P<0.05)]明显减少,而bcl-2[(1.34±0.18)比(0.27±0.08),P<0.05)]水平明显增加。与此相对应,与灭活E.coli处理的细胞相比较,加入SFN(包括SFN组和热灭活E.coli+SFN组),ROS水平明显下降[(1.27±0.17)比(6.30±1.50),P<0.05)],而线粒体膜电位明显上升[(1.18±0.10)比(0.14±0.01),P<0.05)]。结论SFN可有效抑制热灭活E.coli诱导的HeLa细胞调亡、稳定线粒体膜电位、降低ROS、抑制炎性因子的产生。因此,SFN可能作为脓毒血症时保护细胞的药物。Objective To evaluate the cell protective effect of sulforaphane(SFN)in sepsis.Methods HeLa cells were treated with heat-killed Escherichia coli(E.coli)or E.coli plus SFN(E.coli+SFN).Normal cells and cells treated with SFN were set as controls.The proinflammatory cytokines interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)were determined by ELISA.Apoptotic cells were determined by flow cytometry with annexin V and propidium iodide staining.Reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were detected by immunofluorescence microscopy.The cleaved caspase-3,BAX,and bcl-2 were examined by Western blotting.Results Cells treated with the heat-killed E.coli showed increased production IL-1β[(121.13±8.44)pg/mL vs.(36.67±5.82)pg/mL,P<0.05],TNF-α[(123.83±7.32)pg/mL vs.(20.63±1.91)pg/mL,P<0.05],activated caspase-3[(2.28±0.19)vs.(1.01±0.19),P<0.05],BAX[(4.81±0.80)vs.(1.04±0.25),P<0.05],and apoptotic cells[(32.49±3.96)%vs.(12.52±1.37)%,P<0.05],while the level of bcl-2[(0.27±0.08)vs.(0.96±0.18),P<0.05]was markedly reduced.In contrast,cells treated with heat-killed E.coli plus SFN showed significantly lowered levels of proinflammatory cytokines(including IL-1β[(45.41±13.16)pg/mL vs.(121.13±8.44)pg/mL,P<0.05],TNF-α[(30.3±6.0)pg/mL vs.(123.8±7.3)pg/mL,P<0.05]),apoptotic cells[(11.34±2.05%)vs.(32.49±3.96%),P<0.05],and activated caspase-3[(1.01±0.07)vs.(2.28±0.19),P<0.05],and BAX[(2.15±0.14)vs.(4.81±0.80),P<0.05],while bcl-2[(1.34±0.18)vs.(0.27±0.08),P<0.05]was increased.Treatment with SFN also increased MMP[(1.18±0.10)vs.(0.14±0.01),P<0.05]and reduced ROS level[(1.27±0.17)vs.(6.30±1.50),P<0.05].Conclusion Treatment with SFN shows significant inhibition on apoptosis and the generation of proinflammatory cytokines.SFN can also protect mitochondria and reduce the production of ROS.Thus,SFN could be a potential therapeutic agent to protect cells during sepsis.

关 键 词：SULFORAPHANE 细胞凋亡 活性氧 炎症因子 

分 类 号：R459.7[医药卫生—急诊医学]