Piper betle L. Modulates Senescence-Associated Genes Expression in Replicative Senescent Human Diploid Fibroblasts

Abstract

Piper betle (PB) is a traditional medicine that is widely used to treat different diseases around Asian region. The leaf extracts contain various bioactive compounds, which were reported to have antidiabetic, antibacterial, anti-inflammatory, antioxidant, and anticancer effects. In this study, the effect of PB aqueous extracts on replicative senescent human diploid fibroblasts (HDFs) was investigated by determining the expressions of senescence-associated genes using quantitative PCR. Our results showed that PB extracts at 0.4 mg/ml can improve cell proliferation of young (143%), presenescent (127.3%), and senescent (157.3%) HDFs. Increased expressions of PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were observed in senescent HDFs compared to young and/or presenescent HDFs. Treatment with PB extracts modulates the transcriptional profile changes in senescent HDFs. By contrast, expressions of SOD1 increased, whereas GPX1, PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were decreased in PB-treated senescent HDFs compared to untreated senescent HDFs. In conclusion, this study indicates the modulation of PB extracts on senescence-associated genes expression of replicative senescent HDFs. Further studies warrant determining the mechanism of PB in modulating replicative senescence of HDFs through these signaling pathways.

Figure 1

Dose response of PB extracts on proliferation of young, presenescent, and senescent HDFs. a denotes p < 0.05 compared to control young HDFs, b denotes p < 0.05 compared to control presenescent HDFs, and c denotes p < 0.05 compared to control senescent HDFs. Data are presented as mean ± SD (n = 3).

Figure 2

Effects of 0.4 mg/ml PB extracts on antioxidant-associated genes expression of HDFs treated for 24 hours. (a) SOD1, (b) SOD2, (c) CAT, (d) GPX1, (e) CCS, and (f) PRDX6. A denotes p < 0.05 compared to control for young HDFs, B denotes p < 0.05 compared to control for presenescent HDFs, and C denotes p < 0.05 compared to control for senescent HDFs. Data are presented as mean ± SEM (n = 3).

Figure 3

Effects of 0.4 mg/ml PB extracts on stress response genes expression of HDFs treated for 24 hours. (a) FOXO3, (b) TP53, (c) CDKN2A, (d) PAK2, (e) MAPK14, and (f) JUN. A denotes p < 0.05 compared to control for young HDFs, B denotes p < 0.05 compared to control for presenescent HDFs, and C denotes p < 0.05 compared to control for senescent HDFs. Data are presented as mean ± SEM (n = 3).

Figure 4

An illustration on the mechanism of PB extracts in ameliorating replicative senescence of HDFs. PB extracts upregulate SOD1 expression which results in reduced oxidative stress. Reduced oxidative stress spares the expressions of GPX1 and PRDX6, decreases DNA damage, suppresses gene expressions of TP53 and CDKN2A (p16) signaling pathways, promotes cell cycle progression, and thus increases cell proliferation of senescent HDFs.