Pharmacological modulation of histone demethylase activity by a small molecule isolated from subcritical water extracts of Sasa senanensis leaves prolongs the lifespan of Drosophila melanogaster

Abstract

Background: Extracts of Sasa senanensis Rehder are used in traditional Japanese medicine; however, little is known about the underlying mechanisms of their potential health benefits.

Methods: S. senanensis leaves were extracted with subcritical water. An active small-molecule was isolated using reversed-phase high-performance liquid chromatography (HPLC), and identified as 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde or PA). The effects of PA on the activity of histone demethylase, the Drosophila melanogaster lifespan and gene expression in Drosophila S2 cells were investigated.

Results: PA inhibited the activity of Jumonji domain-containing protein 2A (JMJD2A) histone demethylase in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 11.6 μM. However, there was no effect on lysine-specific demethylase 1 (LSD1), histone deacetylase 1 (HDAC1) or HDAC8. PA significantly extended the lifespan of female, but not male, Drosophila. In Drosophila S2 cells, the eukaryotic translation initiation factor 4E binding protein (4E-BP) was up-regulated by PA exposure.

Conclusions: Our findings provide insight into the possible relationship between the pharmacological modulation of histone demethylation and lifespan extension by PA; they might also be important in the development of alternative therapies for age-related disorders.

Figure 1

Purification and identification of PA. (A) Purification of antioxidative compounds from S. senanensis leaves. Subcritical water extracts were prepared from the leaves and fractionated by HPLC with MeOH/H₂O (17:83). The SOSA was assessed for each fraction, and the highest value detected in fraction 4. (B) Purification of the compound by HPLC. Fraction 4 was further fractionated, and fraction 4-II found to contain the highest SOSA. (C) EI-MS spectra data of purified PA, which was identified using the SDBS. (D–F) HPLC chromatograms of fraction 4-II (D), synthetic PA (E), and a mixture of fraction 4-II and synthetic PA (F), in which the two compounds were co-eluted as a single peak. (G) Chemical structure of PA.

Figure 2

Effect of PA on adipocyte differentiation. Inhibition of human subcutaneous preadipocyte differentiation by PA. Differentiation was determined by the amount of lipid accumulation measured by total triglyceride. Data are expressed as a percentage of the control value. Values are mean ± standard error of the mean (SEM; n = 3).

Figure 3

Effects of PA on the activities of histone demethylases. (A) Effects of PA, apocynin or 2,4-PDCA on histone demethylase JMJD2A activity. IC₅₀ values are mean ± SEM of three separate experiments. (B) Effects of PA, apocynin or 2,4-PDCA on histone demethylase JMJD2B activity. IC₅₀ values are mean ± SEM of three separate experiments. (C) Effects of PA, apocynin or 2,4-PDCA on histone demethylase JMJD2C activity. IC₅₀ values are mean ± SEM of three separate experiments. (D) Effects of PA or apocynin on the activities of lysine-specific demethylase LSD1, histone deacetylase HDAC1 and HDAC8. Values are mean ± SEM (n = 4). Data are expressed as a percentage of the control value in each experiment.

Figure 4

View of the conformation of PA docked in the JMJD2A active site. (A) Binding mode of 2,4-PDCA in JMJD2A. (B) Binding mode of PA in JMJD2A.

Figure 5

Effects of PA on the lifespan and egg-to-adult viability of Drosophila. (A) Survival curves of females kept on media containing PA. The mean lifespan of female Drosophila kept on 0.3, 1 and 3 mM PA was increased by 13%, 23% and 13%, respectively (p<0.001; log-rank test). (B) The survival curves of males revealed no effects of PA. (C) The viability of females was not affected at concentrations of up to 10 mM. (D) The viability of males was reduced by PA, even at 1 mM. Larval development was arrested at 100 mM in both genders (p<0.05, t-test).

Figure 6

Effect of PA on gene expression in Drosophila S2 cells. qRT-PCR analysis of 4E-BP or ferrochelatase mRNA in Drosophila S2 cells treated with 100 μM PA for 2 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is included as an internal control. Results are mean ± SEM (n = 5). *p<0.01 compared with vehicle (0.1% DMSO).