摘要
目的探讨迷迭香酸(RA)对过氧化氢(H_(2)O_(2))诱导的视网膜色素上皮细胞(ARPE-19)衰老的作用及可能机制。方法首先用不同浓度(100,200,400,800,1600μmol/L)H_(2)O_(2)处理ARPE-19细胞,CCK-8法检测细胞增殖,筛选合适的H_(2)O_(2)浓度构建细胞衰老模型。然后将ARPE-19细胞分为对照组、H_(2)O_(2)组(200μmol/L)和不同浓度(20,40,80μmol/L)RA预处理+H_(2)O_(2)(200μmol/L)组,CCK-8法检测细胞增殖,筛选最佳RA干预浓度。最后将ARPE-19细胞分为对照组、H_(2)O_(2)组(200μmol/L)、H_(2)O_(2)(200μmol/L)+RA(80μmol/L)组,用倒置显微镜观察各组细胞形态,SA-β-gal染色法检测细胞衰老情况,Western blotting检测衰老标志蛋白P16、P21的表达以及HMGB1,Caveolin-1的表达。结果ARPE-19细胞增殖率随H_(2)O_(2)刺激浓度的增加而降低(各浓度组两两比较,均P<0.01),随着RA浓度的增加而增加(各浓度组两两比较,均P<0.01)。倒置显微镜观察显示,与对照组相比,H_(2)O_(2)组ARPE-19细胞数量明显减少,细胞衰老率明显增加(均P<0.05);与H_(2)O_(2)组相比,H_(2)O_(2)+RA组ARPE-19细胞数量明显增多,细胞衰老率明显降低(均P<0.05)。Western blotting检测显示,H_(2)O_(2)组细胞P16,P21蛋白表达较对照组明显增加(P<0.05);H_(2)O_(2)+RA组细胞P16,P21蛋白表达较H_(2)O_(2)组明显降低(P<0.05);H_(2)O_(2)组细胞HMGB1和Caveolin-1的蛋白表达较对照组明显增加(P<0.05);H_(2)O_(2)+RA组细胞HMGB1和Caveolin-1的蛋白表达较H_(2)O_(2)组明显降低(均P<0.05)。结论RA可以减轻H_(2)O_(2)诱导的ARPE-19细胞衰老,其机制可能与RA抑制HMGB1和Caveolin-1的激活有关。
Objective To investigate the effect of rosmarinic acid(RA)on hydrogen peroxide(H_(2)O_(2))-induced senescence of retinal pigment epithelial cells(ARPE-19)and its possible mechanism.Methods Firstly,ARPE-19 cells were respectively treated with different concentrations of H_(2)O_(2)(100,200,400,800,1600μmol/L),and the cell proliferation was detected by CCK-8 assay to screen the appropriate concentration of H_(2)O_(2)for constructing the cell senescence model.Then,ARPE-19 cells were randomly divided to control group,H_(2)O_(2)group(200μmol/L),and RA(20,40,80μmol/L)pretreatment+H_(2)O_(2)(200μmol/L)groups.CCK-8 assay was used to detect the cell proliferation for screening the optimal intervention concentration of RA.Finally,ARPE-19 cells were randomly assigned to control group,H_(2)O_(2)group(200μmol/L),and H_(2)O_(2)(200μmol/L)+RA(80μmol/L)group.The cellular morphology was observed under an inverted microscope,and the cellular senescence was detected by SA-β-gal staining.Western blotting was used to detect the protein expression levels of P16,P21,HMGB1 and Caveolin-1.Results The proliferation rate of ARPE-19 cells was decreased with the increase of H_(2)O_(2)concentration(pairwise comparison of different concentration groups,all P<0.01),and increased with the increase of RA concentration(pairwise comparison of different concentration groups,all P<0.01).Under an inverted microscope,the number of ARPE-19 cells in H_(2)O_(2)group was significantly decreased and the cell senescence rate was significantly increased compared with control group(all P<0.05).Compared with H_(2)O_(2)group,the number of ARPE-19 cells was significantly increased in H_(2)O_(2)+RA group,and the cell senescence rate was significantly decreased(all P<0.05).Western blotting showed that the protein expression levels of P16 and P21 in H_(2)O_(2)group were significantly higher than those in control group(P<0.05),and the protein expression levels of P16 and P21 in H_(2)O_(2)+RA group were significantly lower than those in H_(2)O_(2)group(P<0.05).The protein expression levels of HMGB1 and Caveolin-1 were significantly higher in H_(2)O_(2)group than in control group(P<0.05),and the protein expression levels of HMGB1 and Caveolin-1 were significantly lower in H_(2)O_(2)+RA group than in H_(2)O_(2)group(P<0.05).Conclusion RA can attenuate H_(2)O_(2)-induced senescence of ARPE-19 cells,which may be related to the inhibition of HMGB1 and Caveolin-1 activation.
作者
关小荣
李蓉
姚国敏
GUAN Xiaorong;LI Rong;YAO Guomin(Department of Ophthalmology,Northwest Polytechnic University Hospital,Xi’an 710072,China;Department of Ophthalmology,First Affiliated Hospital of Xi’an Medical University)
出处
《山西医科大学学报》
CAS
2023年第2期222-228,共7页
Journal of Shanxi Medical University
基金
陕西省科技厅自然科学基金-社发项目(2021SF-157)
陕西省科技厅自然科学基金-面上项目(2021JM-490)
西安市科技局医学研究项目(21YXYJ0127)。
