肌肽对脂多糖诱导脑微血管内皮细胞凋亡的保护作用及机制探讨
|
摘要
目的探讨肌肽对脂多糖(lipopolysaccharides,LPS)引起脑微血管内皮细胞(HBMEC)凋亡的保护作用及机制。方法用倒置显微镜观察细胞形态变化;MTT法检测细胞存活率;流式细胞仪检测细胞内的活性氧(ROS)含量及细胞凋亡情况;Western blot法检测NF-κB P65、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)蛋白表达。结果与对照组相比,LPS组细胞存活率略降低(P>0.05),早期凋亡率明显增加,同时ROS含量、核内NF-κB P65及胞浆IL-1β和TNF-α的含量均显著增加(P<0.05)。与LPS组相比,LPS+肌肽组(10 mmol/L、20 mmol/L、40 mmol/L)凋亡率有明显降低,ROS含量、核内NF-κB P65蛋白量及胞浆IL-1β和TNF-α的含量均不同程度显著降低(P<0.05)。结论肌肽可能通过抑制LPS诱导ROS释放,避免NF-κB P65过度激活,进而减少IL-1β和TNF-α分泌,对脑微血管内皮细胞发挥保护作用。
Objective To investigate the protective effect of carnosine on apoptosis in HBMEC induced by LPS and its mechanisms. Methods The injury model was established by treating HBMEC cells with LPS in vitro. Inverted microscope was used to observe cell morphology. The cell viability was detected by MTT assay, and the cell apoptosis rates were measured by flow cytometry. The reactive oxygen species(ROS) level in cells was detected by fluorescent probe CDFH-DA. The expressions of NF-κB P65, IL-1β and TNF-α were explored by Western blot. Results Compared with control group, the cell viability was slightly decreased in LPS group(P>0.05). The apoptosis rate, the level of ROS, nuclear NF-κB P65, IL-1βand TNF-α increased obviously(P<0.01) in LPS group. Compared with LPS group, the apoptosis rate, the level of ROS, nuclear NF-κB P65, IL-1β and TNF-α reduced obviously(P<0.01) in LPS + carnosine group(10 mmol/L,20 mmol/L,40 mmol/L). Conclusions Carnosine has protective effect on apoptosis of HBMEC cells injury induced by LPS. The mechanism may be related that carnosine inhibited LPS induced ROS production, avoided NF-κB pathway overactivation, and further decreased the secretion of IL-1β and TNF-α.
Objective To investigate the protective effect of carnosine on apoptosis in HBMEC induced by LPS and its mechanisms. Methods The injury model was established by treating HBMEC cells with LPS in vitro. Inverted microscope was used to observe cell morphology. The cell viability was detected by MTT assay, and the cell apoptosis rates were measured by flow cytometry. The reactive oxygen species(ROS) level in cells was detected by fluorescent probe CDFH-DA. The expressions of NF-κB P65, IL-1β and TNF-α were explored by Western blot. Results Compared with control group, the cell viability was slightly decreased in LPS group(P>0.05). The apoptosis rate, the level of ROS, nuclear NF-κB P65, IL-1βand TNF-α increased obviously(P<0.01) in LPS group. Compared with LPS group, the apoptosis rate, the level of ROS, nuclear NF-κB P65, IL-1β and TNF-α reduced obviously(P<0.01) in LPS + carnosine group(10 mmol/L,20 mmol/L,40 mmol/L). Conclusions Carnosine has protective effect on apoptosis of HBMEC cells injury induced by LPS. The mechanism may be related that carnosine inhibited LPS induced ROS production, avoided NF-κB pathway overactivation, and further decreased the secretion of IL-1β and TNF-α.
引文
[1]Tabit CE,Chung WB,Hamburg NM,et al.Endothelial dysfunction in diabetes mellitus:molecular mechanisms and clinical implications[J].Rev Endocr Metab Disord,2010,11(1):61-74.
[2]Liu D,Zhang D,Scafidi J,et al.C1 inhibitor prevents Gram-negative bacterial lipopolysaccharide-induced vascular permeability[J].Blood,2005,105(6):2350-2355.
[3]赵艳,赵颂,李晴,等.肌肽对谷氨酸诱导SH-SY5Y细胞凋亡的保护作用[J].中国生化药物杂志,2015,35(12):34-37.
[4]方超,李晴,鲁美丽,等.肌肽对大鼠脑片缺氧/再灌损伤的保护作用[J].中国生化药物杂志,2015,35(9):41-47.
[5]赵丹婷,刘影,赵艳,等.肌肽对糖尿病大鼠海马中氧化应激及NF-κB信号通路的影响[J].中国临床解剖学杂志,2018,36(5):514-519.
[6]Sun YX,Ge X,Li M,et al.Dyrk2 involved in regulating LPS-induced neuronal apoptosis[J].Int J Biol Macromol,2017,104(Pt A):979-986.
[7]Li C,Ma D,Chen M,et al.Ulinastatin attenuates LPS-induced human endothelial cells oxidative damage through suppressing JNK/c-Jun signaling pathway[J].Biochem Biophys Res Commun,2016,474(3):572-578.
[8]Sun X,Yang Y,Shi J,et al.NOX4-and Nrf2-mediated oxidative stress induced by silver nanoparticles in vascular endothelial cells[J].J Appl Toxicol,2017,37(12):1428-1437.
[9]Ridker PM,Everett BM,Thuren T,et al.CANTOS Trial Group.Antiinflammatory therapy with canakinumab for atherosclerotic disease[J].N Engl J Med,2017,377(12):1119-1131.
[2]Liu D,Zhang D,Scafidi J,et al.C1 inhibitor prevents Gram-negative bacterial lipopolysaccharide-induced vascular permeability[J].Blood,2005,105(6):2350-2355.
[3]赵艳,赵颂,李晴,等.肌肽对谷氨酸诱导SH-SY5Y细胞凋亡的保护作用[J].中国生化药物杂志,2015,35(12):34-37.
[4]方超,李晴,鲁美丽,等.肌肽对大鼠脑片缺氧/再灌损伤的保护作用[J].中国生化药物杂志,2015,35(9):41-47.
[5]赵丹婷,刘影,赵艳,等.肌肽对糖尿病大鼠海马中氧化应激及NF-κB信号通路的影响[J].中国临床解剖学杂志,2018,36(5):514-519.
[6]Sun YX,Ge X,Li M,et al.Dyrk2 involved in regulating LPS-induced neuronal apoptosis[J].Int J Biol Macromol,2017,104(Pt A):979-986.
[7]Li C,Ma D,Chen M,et al.Ulinastatin attenuates LPS-induced human endothelial cells oxidative damage through suppressing JNK/c-Jun signaling pathway[J].Biochem Biophys Res Commun,2016,474(3):572-578.
[8]Sun X,Yang Y,Shi J,et al.NOX4-and Nrf2-mediated oxidative stress induced by silver nanoparticles in vascular endothelial cells[J].J Appl Toxicol,2017,37(12):1428-1437.
[9]Ridker PM,Everett BM,Thuren T,et al.CANTOS Trial Group.Antiinflammatory therapy with canakinumab for atherosclerotic disease[J].N Engl J Med,2017,377(12):1119-1131.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |