Screening SIRT1 Activators from Medicinal Plants as Bioactive Compounds against Oxidative Damage in Mitochondrial Function

Sirtuin type 1 (SIRT1) belongs to the family of NAD+ dependent histone deacetylases and plays a critical role in cellular metabolism and response to oxidative stress. Traditional Chinese medicines (TCMs), as an important part of natural products, have been reported to exert protective effect against oxidative stress in mitochondria. In this study, we screened SIRT1 activators from TCMs and investigated their activities against mitochondrial damage. 19 activators were found in total by in vitro SIRT1 activity assay. Among those active compounds, four compounds, ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A, were further studied to validate the SIRT1-activation effects by liquid chromatography-mass spectrometry and confirm their activities against oxidative damage in H9c2 cardiomyocytes exposed to tert-butyl hydroperoxide (t-BHP). The results showed that those compounds enhanced the deacetylated activity of SIRT1, increased ATP content, and inhibited intracellular ROS formation as well as regulating the activity of Mn-SOD. These SIRT1 activators also showed moderate protective effects on mitochondrial function in t-BHP cells by recovering oxygen consumption and increasing mitochondrial DNA content. Our results suggested that those compounds from TCMs attenuated oxidative stress-induced mitochondrial damage in cardiomyocytes through activation of SIRT1.


Introduction
Sirtuin type 1 (SIRT1) belongs to the family of class III histone deacetylases (HDAC) that consume NAD + during deacylation cycle. It has been reported that, in mammals, SIRT1 plays a critical function in cellular metabolism and response to oxidative stress [1][2][3][4].
Recently, researchers have found that SIRT1 activators can protect mitochondrial function from oxidative-induced mitochondrial damage in various types of cell through regulating PGC-1 and multiple transcription factors [5][6][7][8][9], which are tightly related to mitochondrial biogenesis and metastasis [10,11]. It is also reported that activators of SIRT1, such as resveratrol [12], can extend lifespan and regulate metabolic disorders [13][14][15]. Therefore, SIRT1 activators exhibit unique pharmacological potentials for treating mitochondrial dysfunction related diseases. Meanwhile, several clinical trials of SIRT1 activators such as SRT1720 for type 2 diabetes and obesity are under way [16].
Natural products have historically been regarded as an important resource of therapeutic agents in pharmaceutical discovery over the past century [17]. Traditional Chinese medicines (TCMs), as an important part of natural products, are mainly governed by empirical experience and fundamental theories such as the Yin and Yang concept [18]. TCMs with Qi Tonification effects including Astragalus membranaceus [19,20], Panax ginseng [21,22], and Panax notoginseng [23,24] have been reported to exert protective effect against oxidative stress in mitochondria. Several compounds isolated from TCMs are reported to regulate SIRT1 activity [25]. However, a comprehensive screening of SIRT1 activators from TCMs has not yet been performed to investigate their protective effects on mitochondrial function against oxidative stress.

Oxidative Medicine and Cellular Longevity
The aim of present study is to discover SIRT1 activators from TCMs and validate their activities against mitochondrial damage. A sensitive in vitro assay to screen SIRT1 activators was performed to discover bioactive compounds from TCMs, and the lead compounds were validated by liquid chromatography-mass spectrometry (LC-MS) analysis. Effects of identified SIRT1 activators on mitochondrial function were further investigated in cardiomyocytes exposed to tert-butyl hydroperoxide (t-BHP). ATP content, intracellular ROS formation, and activity of Mn-SOD were measured. Moreover, oxygen consumption and mitochondrial DNA content of cardiomyocytes were used to evaluate the effects of those SIRT1 activators on mitochondrial function.

Fluorescent Probe Based Assay for SIRT1 Modulation
Effects of Compounds. The measurement of SIRT1 activity and effects of compounds on SIRT1 activation were performed by a previously reported fluorescent method [26]. Briefly, SIRT1 was incubated with a TPE-GK(Ac)YDD probe (20 M) in the presence of the tested compound (50 M). Fluorescence intensity was recorded by a TECAN infinite F200 microplate reader with excitation wavelength 320 nm and emission wavelength 465 nm.
A total of 195 constituents of TCMs were screened by the assay to evaluate their regulatory effects on SIRT1 activity. The detailed information related to the chemicals and their CAS number were illustrated in Supplementary Material available online at http://dx.doi.org/10.1155/2016/4206392. The inhibition or activation of SIRT1 was calculated by the following equation: and represented the fluorescence intensity of tested sample group with various test compounds and control group without test compounds. 0 and 0 represented the fluorescence intensity of tested sample and control when incubated without SIRT1 protein. To explore the dose-related effects of these compounds, several active compounds, including ginsenoside Rb 2 , ginsenoside Rc, and schisandrin A, were further tested with the concentrations of 1, 10, 25, and 50 M.

Validation of SIRT1 Activators by LC-MS Analysis.
In order to validate activation of SIRT1, SIRT1 (10 g/mL) was added to TPE-GK(Ac)YDD (20 M) and NAD + (Sigma, 3 mM) for 1 h incubation at 37 ∘ C in the presence or absence of ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A (50 M), respectively. Samples were boiled at 100 ∘ C for 10 min to degenerate SIRT1 and terminate reaction.

Measurement of ATP Content and Intracellular ROS.
H9c2 cells were seeded in 96 wells at the density of 4,000/well. The cells were preincubated with ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A (20 M) for 24 h before being exposed to t-BHP (300 M) for 1 h. Intracellular ATP content was measured by CellTiter-Glo Ⓡ Luminescent Assay kit (Promega) according to the instruction of manufacture. Intracellular ROS content was measured by DCFH-DA probe (5 M) whilst fluorescence intensity was recorded by a TECAN infinite F200 Multifunction microplate with excitation wavelength 485 nm and emission wavelength 535 nm. The changes of ATP content and ROS accumulation were calculated by comparing the luminescent or fluorescent signal of the treated cells with that of untreated H9c2 cells.

Detection of Mn-SOD Activity.
Manganese superoxide dismutase (Mn-SOD) was an antioxidative enzyme, which protected against ROS-induced damage [27]. To measure Mn-SOD activity, H9c2 cells were seeded in 6-well plate in the density of 4 × 10 5 /mL. H9c2 cells were preprotected for 24 h by ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A (20 M) before being exposed to t-BHP (300 M) for 1 h. The cells were lysed and the concentration of total protein was measured by BCA assay kit. Mn-SOD activity in total protein was measured by Mn-SOD assay kit (Beyotime, China).

Oxygen Consumption
Assay. 2 × 10 6 H9c2 cells were seeded in 100 mm culture plate. After being grown to stable attachment, cells were preincubated with ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A at final concentrations (20 M) for 18 h before being exposed to t-BHP (100 M) for 1 h. Cells were washed with PBS twice, subsequently collected by trypsinization followed by centrifugation, and resuspended in fresh medium. Respiratory activity was measured with a Clark-type oxygen electrode (Oxytherm, Hansatech Instruments, UK). An aliquot (1 mL) of suspended cells (2 × 10 6 cells/mL) was placed in the airtight liquid-phase oxygen electrode chamber. The system was maintained at 37 ∘ C. After equilibration, the slope of oxygen consumption in H9c2 cells was measured. Every 1 × 10 6 cells oxygen consumption was calculated as the basic respiration rate of each group. The experiment was also performed in the presence of SIRT1 inhibitor, EX527 at final concentration of 20 M to investigate whether the effect of ginsenoside Rb 2 can be prevented by SIRT1 inhibitor.

Measurement of Mitochondrial DNA Content.
Real-time PCR was used to determine relative quantities of mitochondrial DNA content in H9c2 cells exposed to t-BHP and cells incubated with SIRT1 activators. Cells were preincubated for 24 h with ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A at final concentrations (20 M) before being exposed to t-BHP (300 M) for 1 h. Cells in normal condition were used as control group. Total DNA was extracted using Mammalian Genomic DNA Miniprep Kits (Sigma, USA). DNA was quantified by measuring 260 values, and 50 ng of total DNA was used for PCRs by GenElute™ QuantiFast SYBR Green PCR Kit (QIAGEN, Germany). Primers specific to the mitochondrial-encoded Atp6 gene (Fw: 5 -ATT ACG GCT CCT GCT CAT A-3 ; Rev: 5 -TGG CTC AAC CAA CCT TCT A-3 ) were used to assess mitochondrial DNA copy numbers. Primers designed against the nuclear-encoded Rpl13 gene (Fw: 5 -CAC AAG AAA ATG GCA CGC AC-3 ; Rev: 5 -GAG CAG AAG GCT TCC TGG G-3 ) were used for normalization.
values were obtained automatically. The number of mitochondrial genes was calculated by 2 −ΔΔ method.

Statistical Analysis.
All values were expressed as the means ± SD. One way ANOVA was used to analyze differences among groups. Statistical analysis was performed using GraphPad Prism. values of less than 0.05 were considered statistically significant.
Representative compounds were chosen to validate their activation effects on SIRT1 activity. Ginsenoside Rb 2 showed 8% to 152% activation with the concentration range of 1∼ 50 M. Ginsenoside Rc exerted 88% activation at the concentration of 50 M. Schisandrin A showed 28% activation at the concentration of 50 M.

Validation of SIRT1 Activators by LC-MS Analysis.
To validate the SIRT1-activation effect of the compounds, liquid chromatography-mass spectrometry (LC-MS) analysis was employed. The specific substrate of SIRT1, TPE-GK(Ac)YDD (Figure 2(a)), was incubated with SIRT1 (10 g/mL) and NAD + (3 mM) for 1 h. The reaction product was identified by LC-MS based on the molecular weight. As shown in Figure 2(b), the deacetylated peptide, TPE-GKYDD, was detected with a loss of Ac (43 Da). When analyzing the deacetylated reaction in the presence of SIRT1 activators, including ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A, the peaks of deacetylated product were significantly elevated (Figure 2(c)). Our findings indicated that these compounds activated SIRT1 in enzymatic reaction.

Effects of SIRT1 Activators on Cardiomyocytes Oxygen
Consumption. The effects of SIRT1 activators on mitochondrial function were further investigated by measuring cellular respiration in H9c2 cells. As shown in Figure 3(a), basal respiration of t-BHP treated cardiomyocytes was significantly dropped comparing with control group. Preincubation with ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A attenuated the decrease of oxygen consumption. Figure 3(b) showed the representative oxygen consumption slope of normal cells (1.65 ± 0.31 nmol O 2 /mL/min), t-BHP treated cells (1.10 ± 0.25 nmol O 2 /mL/min), and ginsenoside Rb 2 treated cells (1.43 ± 0.25 nmol O 2 /mL/min). The results suggested that these SIRT1 activators recovered the oxygen consumption rate in t-BHP injured cardiomyocytes. In the presence of SIRT1 inhibitor EX527 [28,29], the protective effect of ginsenoside Rb 2 was blocked, which indicated that the effect of ginsenoside Rb 2 to recover the oxygen consumption rate was SIRT1 dependence (Figure 3(c)).

Effects of SIRT1 Activators on ATP Content and ROS Accumulation.
As a specific parameter of mitochondrial function, intracellular ATP contents in cardiomyocytes exposed to oxidative stress were measured. After cells were exposed to t-BHP for 1 h, the content of ATP was significantly decreased. As shown in Figure 4, preprotection of cells by ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A led to the recovery of ATP content, suggesting that those SIRT1 activators reversed the decreased mitochondrial energy metabolism induced by t-BHP. Mitochondrial oxidative stress was often caused by increased intracellular ROS formation. Figure 5 showed that the intracellular ROS was significantly increased after t-BHP treatment. Pretreatments of SIRT1 activators, ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A, kept intracellular ROS levels on the normal condition.

Effects of SIRT1 Activators on Mn-SOD Activity.
Mn-SOD was one of the antioxidative enzymes in mitochondria that assured mitochondrial oxidative stress resistance. As shown in Figure 6, when cells were exposed to t-BHP (300 M) for 1 h, activity of Mn-SOD was decreased. Preprotection of cells by ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A enhanced the activity of Mn-SOD compared with t-BHP group.

Effects of SIRT1 Activators on Mitochondrial DNA Content.
To verify the effect of ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A of mitochondrial protection or biogenesis, cells were injured by t-BHP after compounds preprotection, and then mitochondrial DNA content was analyzed. As shown in Figure 7, t-BHP treatment reduced mitochondrial DNA content compared to control group. Pretreatments of ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, or schisandrin A significantly elevated the mitochondrial DNA content. Our findings suggested that these natural SIRT1 activators facilitated mitochondrial biogenesis.

Discussion
In the present study, we screened and identified 19 SIRT1 activators, such as ginsenoside Rg 3 , ginsenoside Rb 2 , ginsenoside Oxidative Medicine and Cellular Longevity     Mitochondrial DNA (versus control) * * * * * * Figure 7: Effects of SIRT1 activators on mitochondrial DNA content. Each bar represented the mean ± SD of triplicate experiments. Compared with t-BHP group, * < 0.05 and * * < 0.01. Rb 3 , ginsenoside F1, and ginsenoside Rc from Panax ginseng, ophiopogonin D from Ophiopogon japonicas, and schisandrin A and schisandrin B from Schisandra chinensis. Interestingly, those herbs consisted of a traditional Chinese formula named Shengmai San, which have been clinically used for the treatment of coronary heart diseases [30,31] and heart failure [32,33]. Four SIRT1 activators from Shengmai San, including ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A, were validated by LC-MS analysis and we found their effects against mitochondrial oxidative damage in further study. Our findings were in accordance with previous reports on other cell lines. Ginsenoside Rc was reported to suppress oxidative stress in HEK293T cells [34], whilst schisandrin A inhibited apoptosis induced by H 2 O 2 in intestinal epithelial cells [35].
Mitochondrial dysfunction has been one of mechanisms in organ injuries and diseases due to its influence on ATP formation, metabolism, and apoptosis [36]. Our findings indicated that those SIRT1 activators elevated ATP content, prevented ROS formation, and increased the activity of Mn-SOD. Mitochondrial DNA content and oxygen consumption were also moderated by SIRT1 activators. Those results indicated that SIRT1 activators protected mitochondrial function by improving mitochondrial DNA content, which led to promotion of ATP content, mitochondrial oxygen consumption, and reduction of ROS formation.

Conclusion
In summary, we identified 19 SIRT1 activators from TCMs. Four active compounds, ginsenoside Rb 2 , ginsenoside F1, ginsenoside Rc, and schisandrin A, exerted significant activities against t-BHP induced oxidative damage in cardiomyocytes. Our findings provided useful evidence to illustrate the cardioprotective effects of TCMs with Tonification effects and led to a new insight into the scientific illustration of TCMs theory.