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[摘要]
目的 探讨欧亚旋覆花总黄酮(IBFTF)对糖基化终末产物(AGEs)诱导小鼠皮肤成纤维细胞株L929衰老的保护作用,和AGEs特异性受体(RAGE)的表达机制。方法 采用0.100 g/L AGEs培养液刺激离体培养的L929细胞48 h制备衰老模型,药物组分别给予不同质量浓度IBFTF (0.1、0.2、0.4 g/L)继续作用6 h,阳性对照组给予0.1 g/L氨基胍半硫酸盐(AG)继续作用6 h,对照组给予完全培养液。检测胞内β-半乳糖苷酶阳性染色细胞数量和细胞周期比例等衰老指标;检测胞内活性氧(ROS)、超氧化物歧化酶(SOD)和丙二醛(MDA)水平等氧化应激指标。采用细胞免疫荧光检测RAGE荧光强度,Western blotting和qRT-PCR分别检测RAGE蛋白和mRNA表达情况。结果 IBFTF可显著抑制AGEs诱导的L929细胞衰老并降低RAGE的蛋白和mRNA表达水平;不同质量浓度IBFTF作用于衰老的L929细胞后,可显著提高细胞内SOD活性,显著降低MDA和ROS水平,具有浓度依赖性。结论 IBFTF对AGEs诱导衰老的L929细胞有保护作用,其机制可能是通过抑制RAGE表达来实现的。
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[Abstract]
Objective To investigate the protective effect of Inula britannica flower total flavonoids (IBFTF) on aging L929 cells induced by advanced glycation end products (AGEs), and to explore the mechanism of the expression of receptor advanced glycation end products (RAGE).Methods The senescence L929 cells model was established by using 0.100 g/L AGEs for 48 h culture in vitro, and then different concentration of IBFTF (0.1, 0.2, and 0.4 g/L) was given to the treatment group for 6 h culture, and 0.1 g/L aminoguanidine hemisulphate (AG) was given to the positive group for another 6 h culture, and while blank group was cultured with common culture medium. Senescence aging index of β-galactosidase staining cell numbers and cell cycle analysis in L929 cells were measured. The levels of reactive oxygen species associated with oxidative stress in the cells, and aging indicators such as superoxide dismutase (SOD), malondialdehyde (MDA) were also examined. The fluorescent intensity of RAGE was detected by immunofluorescence. The expression of RAGE protein and mRNA was detected by Western blotting and qPCR, respectively.Results IBFTF significantly inhibited AGEs-induced L929 cells senescence and decreased RAGE protein and mRNA expression. Different concentrations of IBFTF could significantly increase the SOD activity and reduce the MDA content and eliminate the reactive oxygen species in aging L929 cells in a dose-dependent manner.Conclusion Protective effect of IBFTF on aging L929 cells induced by AGEs may be related to its effect on the surpressing the expression of RAGE.
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[基金项目]
重庆市卫生局中医药科技资助项目(ZY20132123)