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. 2017:2017:2391820.
doi: 10.1155/2017/2391820. Epub 2017 Jul 2.

The Preconditioning of Berberine Suppresses Hydrogen Peroxide-Induced Premature Senescence via Regulation of Sirtuin 1

Xiaofei Zhu  1   2   3 , Haodi Yue  1 , Xiaofang Guo  4 , Jingyi Yang  1 , Jingshuo Liu  1 , Jiangtao Liu  1 , Ruijie Wang  1 , Wenjuan Zhu  1
Affiliations

The Preconditioning of Berberine Suppresses Hydrogen Peroxide-Induced Premature Senescence via Regulation of Sirtuin 1

Xiaofei Zhu et al. Oxid Med Cell Longev. 2017.

Abstract

With a long history of application in Chinese traditional medicine, berberine (BBR) was reported to exhibit healthspan-extending properties in some age-related diseases, such as type 2 diabetes and atherosclerosis. However, the antiaging mechanism of BBR is not completely clear. By means of hydrogen peroxide- (H2O2-) induced premature cellular senescence model, we found that a low-concentration preconditioning of BBR could resist premature senescence in human diploid fibroblasts (HDFs) measured by senescence-associated β-galactosidase (SA-β-gal), accompanied by a decrease in loss of mitochondrial membrane potential and production of intracellular reactive oxygen species (ROS). Moreover, the low-concentration preconditioning of BBR could make cells less susceptible to subsequent H2O2-induced cell cycle arrest and growth inhibition. Experimental results further showed that the low concentration of BBR could induce a slight increase of ROS and upregulate the expression level of sirtuin 1 (SIRT1), an important longevity regulator. H2O2-induced activation of checkpoint kinase 2 (Chk2) was significantly attenuated after the preconditioning of BBR. The present findings implied that the low-concentration preconditioning of BBR could have a mitohormetic effect against cellular senescence triggered by oxidative stress in some age-related diseases through the regulation of SIRT1.

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Figures

Figure 1
Figure 1
Antisenescence effect of BBR on H2O2-induced premature senescence in human diploid fibroblasts by β-galactosidase staining. 2BS cells were pretreated with different concentrations of BBR for 12 hr before exposure to 200 μmol/L H2O2. For senescence assay, cells were cultured for four days with new fresh DMEM medium. On the fifth day, the blue precipitate can be seen by X-gal dye in senescent cells as the Materials and Methods mentioned. (a), (b), and (c) Pretreatment with 6, 12, and 20 μmol/L BBR, respectively. (d) H2O2 treatment alone. (e) Untreated control. (f) The positive rates of SA-β-gal were calculated by counting the blue-dyed cells with a total of 200 cells at each visual field. ∗p < 0.05, ∗∗p < 0.01. The results are representative of three separate experiments.
Figure 2
Figure 2
Cell cycle arrest and protein expression of H2O2-induced premature senescence in human diploid fibroblasts. 2BS cells were exposed to a sublethal concentration of 200 μmol/L H2O2, or pretreated with 12 μmol/L BBR for 12 hr before exposure. (a), (b) The influence of BBR in H2O2-induced cell cycle distribution. (c) The changes of SIRT1 expression and phosphorylation of Chk2 in H2O2-exposed human diploid fibroblasts. (d) Relative expression levels of Sirt1 and phospho-Chk2 in H2O2-exposed human diploid fibroblasts by gray analysis. ∗p < 0.05, ∗∗p < 0.01. The results are representative of three separate experiments.
Figure 3
Figure 3
Protective effect of BBR on H2O2-induced mitochondrial membrane potential (Δψm) loss in human diploid fibroblasts. 2BS cells were pretreated with or without 12 μmol/L BBR for 12 hr before a 2 hr exposure to 200 μmol/L H2O2 and then were stained with Rhodamin123. (a) The diagram from flow cytometry test. M1 represents the percentage of Δψm loss. (b) Histogram of the mean fluorescence intensity of M1. ∗p < 0.05, ∗∗p < 0.01. The results are representative of three separate experiments.
Figure 4
Figure 4
Protective effect of BBR on H2O2-induced intracellular ROS production in human diploid fibroblasts. 2BS cells were pretreated with or without 12 μmol/L BBR prior to a 2 hr exposure of 200 μmol/L H2O2, or treated with BBR alone as the Materials and Methods mentioned. (a) The diagram of fluorescence DCF detection with different treatments from flow cytometry analysis. (b) Histogram of the mean fluorescence intensity of DCF, reflecting the percentages of ROS generation in different treatment group. ∗∗p < 0.01. The results are representative of three separate experiments.
Figure 5
Figure 5
Protective effect of BBR on H2O2-induced reduction of SIRT1 expression and augment of phosphorylation of Chk2 in human diploid fibroblasts. 2BS cells were pretreated with 12 μmol/L BBR for 12 hr before a 2 hr or 4 hr exposure to 200 μmol/L H2O2. (a) Sirt1 and phospho-Chk2 was detected by western blotting. (b) Relative expression levels of Sirt1 and phospho-Chk2 in cells with different treatment by gray analysis. ∗p < 0.05, ∗∗p < 0.01. The results are representative of three separate experiments.
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