Resveratrol and Quercetin Potentiate the Cell Protection and Rescue Effects of NAD + Precursors in HEK293 Cells Challenged by DNA Damaging Agent,N -Methyl- N′ -nitro- N -nitrosoguanidine

Abstract

DNA damage plays an essential role in the human ageing process. Recently, accumulating evidence has demonstrated that a decreased level of nicotinamide adenine dinucleotide (NAD⁺) is involved in human ageing and suggested that the natural supplements of NAD⁺ precursors and its homeostasis regulators might serve as a promising modality to slow down the human ageing process. In the present study, we analyzed the combinational effects and potential mechanism of NAD⁺ precursors, nicotinic acid (NA) and nicotinamide (NM), and the NAD⁺’ homeostasis regulators, resveratrol (R), and quercetin (Q) in the protection and rescue of HEK293 cells from N-methyl-N'-nitro-N nitrosoguanidine (MNNG)-induced DNA damage. The results indicate that resveratrol and quercetin can significantly potentiate the cell protection and rescue effects of NAD⁺ precursors in HEK293 cells attacked by the DNA damaging agent, MNNG. Intracellular NAD⁺ homeostasis and the PARP1 activation status are the key factors in determining the fate of the cells under DNA damaging stress.

Abbreviations

NAD⁺, nicotinamide adenine dinucleotide; SIRTs, sirtuin family enzymes; PARPs, poly(ADP-ribose) polymerases; MNNG, N-methyl-N'-nitro-N nitrosoguanidine; NMN, nicotinic acid mononucleotide; NA, nicotinic acid; NM, nicotinamide; Q, quercetin; R, resveratrol; QA, quinolinic acid.

Introduction

Nicotinamide adenine dinucleotide (NAD⁺) is a critical coenzyme that is involved in many essential biological processes in live cells, such as adenosine triphosphate (ATP) production, DNA repair, epigenetic modulation of gene expression, intracellular calcium signaling, and immune regulatory functions. ¹ In energy metabolism, NAD⁺ and its reduced form (NADH) act as electron and proton carrier molecules cycling back and forth in multiple metabolic pathways, such as glycolysis, β-oxidation, and oxidative phosphorylation. ² Besides, its crucial role in redox reactions, NAD⁺ also functions as a critical or exclusive substrate for many important enzymes, such as the sirtuin enzymes family (SIRTs), poly(ADP-ribose) polymerases (PARPs), and cyclic ADP-ribose synthases.^3‐5 Recently, there has been a surge of interest in studying the biology of NAD⁺ precursors due to an accumulative body of evidence indicating a direct correlation between the declined NAD⁺ level and many human diseases, such as diabetes, obesity, neurodegenerative diseases, and human ageing.^6‐13

It is well-understood that in mammals, the intracellular levels of NAD⁺ are determined by its biosynthesis and biodegradation activities. The biosynthetic pathways of NAD⁺ can be classified into the de novo and salvage pathways.^1,2 In the de novo pathway, the precursors of NAD⁺ include nicotinic acid (NA), nicotinic acid riboside (NAR), and tryptophan (TRYP). Through either a single or multiple metabolic process, these precursors are all converted into a common intermediate metabolite, termed nicotinic acid mononucleotide (NAM). The conversion of NA and NAR to NAM is mediated by nicotinic acid phosphoribosyltransferase ¹⁴ and nicotinamide kinase (NRK), ¹⁵ respectively. TRYP is first converted to quinolinic acid via the kynurenine pathway, ¹⁶ and then to NAM by quinolinic acid phosphoribosyltransferase. ¹⁷ The resulting NAM is then catalyzed to nicotinic acid adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase (NMNATs), and finally to NAD⁺ by glutamine-dependent NAD⁺ synthetases.^18,19

In the salvage NAD⁺ synthesis pathway, the precursor molecule, nicotinamide (NM), is generated by NAD⁺ degradation when acting as a substrate of multiple enzymes, such as SIRTs, PARPs, and cyclic adenosine 5′-diphosphate ribose (cADPRs). NM is converted back to NAD⁺ by a two-step reaction, first to nicotinamide mono nucleotide (NMN) by nicotinamide phosphoribosyltransferase, and then NMN to NAD⁺ by NMNAT. ²⁰ Besides NM and NMN, nicotinamide riboside is another recently identified NAD⁺ precursor in the NAD⁺ salvage pathway, where it is first converted to NMN by NRK, and then from NMN to NAD⁺ by NMNAT. ²¹

In the ageing process, declined cellular and tissue NAD⁺ levels were frequently observed in many organisms, including human beings.^12,13,22,23 The mechanistic basis for NAD⁺ decline in ageing was proposed to be primarily related to two pathological conditions, i.e., excessive DNA damage caused by increased oxidative stress, and excessive cytokine production caused by chronic inflammation. ¹ In the first condition, in response to excessive DNA damage, PARPs are hyper-activated, resulting in the depletion of the cellular pool of NAD⁺. In the second condition, excessive cytokine production causes hyper-activation of CD38, leading to cellular NAD⁺ depletion. One critical consequence of the decline of intracellular NAD⁺ is the significant compromising of many important enzymatic biological processes that use NAD⁺ as substrate, e.g. with the sirtuin family proteins, the most notable enzymes that are frequently linked with extended life spans in many mammals. ²⁴

Given that most of the previous NAD⁺ research works were mostly focusing on either a single NAD⁺ precursor or a single NAD⁺ regulatory factor, it is worthwhile to explore the integrated effects by modulating NAD⁺ homeostasis from these two aspects. The present research, therefore, was specially designed to evaluate the combinational effects of NAD⁺ precursors, NA and NM, which respectively sit in the de novo and the salvage NAD⁺ biogenesis pathways, with resveratrol and quercetin, the two intensively studied phytocompounds with NAD⁺ homeostasis modulatory activities.^24‐26 Intracellular NAD⁺ boosting effects were also compared between the NA + NM combinational approach and the NMN alone approach.

Methods

Cell Line and Phytocompounds

Human embryonic kidney cell line, HEK293 was obtained from the American Type Culture Collection. High purity (over 98%) NA, NM, NMN, resveratrol, and quercetin were gifts from Dr. Ma's Laboratories Inc. (Vancouver, Canada).

Cell Viability Assay

HEK293 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin in a CO₂ (5%) incubator at 37°C. For cell viability assay, cells were plated into 96-well plates (Sarstedt) at a density of 5  ×  10³ cells/well overnight. On the following day, cells were treated with different test compounds and the indicated combinations, followed by incubation in a CO₂ incubator for 24, 48, and 72 h. At the indicated time points, cell viability was determined using water-soluble tetrazolium salt-1 (WST-1) kit (Alkali Scientific Inc) according to the manufacturer's instructions. The percentage of cell viability relative to control was calculated with the following formula: ([A450 of the experiment − A450 of background]/[A450 of control − A450 of background])*100.

Evaluation of Cell Protective Effects of the Test Compounds From DNA Damage

HEK293 cells were plated in 96-well plates as described earlier and treated on the following day with different compounds and their combinations, as described above, for 24 h. Cells were then exposed to MNNG at 3 different concentrations (10, 50, or 100 μM) for 1 h, followed by a medium exchange with complete DMEM. Cell viability was determined after 48 h, as described above.

Evaluation of Cell Rescue Effects of the Test Compounds and Their Combinations from MNNG-Induced DNA Damage

HEK293 cells were plated in 96-well plates, as described above, and pretreated on the following day with MNNG (100 μM) for 1 h, followed by media exchange with the indicated compounds and their combinations, and incubated for 48 and 72 h. Cell viability was determined as described above.

Immunofluorescence Assay for DNA Damage Foci Detection

HEK293 cells were plated in 24 well plates with coverslips at a density of 5 ×  10⁴ cells per well. On the following day, DNA damage was induced by MNNG treatment at a concentration of 10 μM for 1 h, followed by media exchange with complete DMEM in the control wells, and with complete DMEM containing NA (5 mM), NM (5 mM), Q (100 μM), R (100 μM) and their indicated combinations in the treatment wells. After incubating for 48 h, DNA damage detection was measured as described earlier. ²⁷ Briefly, cells were washed with phosphate-buffered saline (PBS) and fixed with cold methanol for 10 min. For gamma-H2AX visualization, cells were fixed with 4% paraformaldehyde in tris-buffered saline (TBS) (50 mM Tris-Cl, pH 7.5, 150 mM NaCl), and then permeabilized with methanol for 1 min on ice. Fixed cells were then blocked with blocking buffer (2% Bovine Serum Albumin (BSA), 0.2% Tween-20 in TBS) for 30 min, followed by primary antibody incubation for overnight and secondary antibody incubation for 1 h. Finally, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL) for 30 min and washed 3 times. The images were acquired using confocal fluorescence microscopy.

Western Blotting and Quantification

HEK293 cells were plated in 6 well plates at a density of 5 ×  10⁵ cells per well. On the following day, DNA damage was induced by MNNG treatment at a concentration of 10 μM for 1 h. Culture media were then exchanged with complete DMEM, NA (5 mM), NM (5 mM), Q (100 μM), R (100 μM), and indicated combinations. Following incubation for 48 hours, all treated and control cells were lysed with lysis buffer (50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton x-100, 0.5% deoxycholic acid sodium salt [w/v]) supplemented with 2 μL/mL protease inhibitor cocktail (Sigma), incubated on ice for 30 min and centrifuged for 10 min to remove cell debris. Cell lysates were then loaded onto SDS–polyacrylamide gel and immunoblotted with respective antibodies (beta-actin [Cell Signaling Technology, Cat. 3700], gamma-H2AX [Abcam, Cat. ab81299] and PARP1 [Santa Cruz Technology, Cat. C2707]). The bands were detected with enhanced chemiluminescence (ECL) reagent (Thermo Scientific). Western blotting signal intensities were quantified using Image J software and expressed as values relative to beta-actin loading control.

Measurement of NAD⁺ Levels

NAD⁺ levels were measured using NAD⁺/NADH Quantification Colorimetric kits (Biovision Inc., America, Cat# K337-100), by following the manufacturer's instructions. Briefly, HEK293 cells were treated with NMN (5 mM) and NA and NM combination (5 mM for both) for 24 h, and the cells were collected in 400 μL of NAD⁺/NADH extraction buffer. Samples from each treatment group were split into two sets, the first of which was used to carry out a thermal decomposition assay of NAD⁺ followed by the enzymatic cycling assay to determine NADH content. The other set was used to measure the total NAD (NADH plus NAD⁺) content by performing the cycling assay without thermal NAD⁺ decomposition. The final products were read at OD₄₅₀ and the values were converted to absolute NAD concentrations based on the simultaneously generated NAD standard curve. The NAD⁺/NADH ratio was calculated as the percentage of (total NAD—NADH)/NADH.

Statistical Analysis

Statistical analysis was performed by Student's unpaired t-test to identify significant differences unless otherwise indicated. Differences were considered significant at P < .05.

Results

NA + NM Combination Approach Significantly Promoted HEK293 Cell Proliferation

The HEK293 cell line was used to test the effect of each individual compound and their combinations on HEK 293 cell growth. Cells were treated with 5 mM NA and NM, and 100 μM R and Q, and, at 24, 48, and 72 h post-incubation, cell viability was determined. The results demonstrated that none of the compounds and their tested combinations caused any cell growth inhibition. Instead, cell proliferation-promoting effects were observed across these treatments (Figure 1A). Among all the test compounds and their varied combinations, NA + NM demonstrated the most significant cell proliferation stimulatory effect at all the three-time points, while NM, NA + NM + Q, and NA + NM + R only showed significant cell growth stimulation effects at relatively later time points. Interestingly, NM treatment alone showed a similar level in stimulating cell proliferation, compared to the combinational treatments, indicating the central role played by NM in stimulating cell proliferation.

Figure 1.

Effects of NA, NM, resveratrol, and quercetin, individually and in combinations, in the stimulation of HEK293 cell proliferation, and protecting and rescuing cells from MNNG-induced cell death. (A) HEK293 cells were plated in 96-well plates (5 × 10³ cells per well) and treated with complete DMEM, NA (5 mM), NM (5 mM), R (100 μM), Q (100 μM), NA + NM, NA + NM + Q and NA + NM + R for 24, 48, or 72 hours. Cell viabilities were determined with WST-1 assay in triplicate experiments. (B) HEK293 cells were plated in 96-well plates (5 × 10³ cells per well) and pretreated with complete DMEM, NA + NM, NA + NM + Q, and NA + NM + R for 24 h. Cells were then treated with MNNG (10, 50, or 100 μM) for 1 hour, followed by medium replacement with complete DMEM. Cell viabilities were measured by WST-1 assay at the 48 h time point post-MNNG treatment. (C) HEK293 cells were pretreated with MNNG (100 μM) for 1 h followed by medium replacement with indicated agents and continuously incubated for 48 and 72 h. Cell viabilities were measured by WST-1 assay. (D) HEK293 cells were pretreated with MNNG (100 μM) for 1 h, followed by medium replacement with complete DMEM, NA + NM, NA + NM + R, or NA + NM + Q and continuously incubated for 48 and 72 h. Cell viabilities were measured by WST-1 assay. All samples were tested in triplicates. Data are shown as the mean ± SD. ^∗P < .05; ^∗∗P < .01; ^∗∗∗P < .001.

Both NA + NM + Q and NA + NM + R Combination Approaches Significantly Protected HEK293 Cells From MNNG-Induced Cell Death

In the present study, we also evaluated the cell-protective effects of NA + NM, NA + NM + R, and NA + NM + Q combinations on HEK293 cells post-MNNG exposure. MNNG is a strong DNA alkylating agent with the capability to cause single and double-strand DNA breaks (DSBs), and, if left un-repaired, results in cell death. ²⁸ HEK293 cells were first treated with NA + NM, NA + NM + R, and NA + NM + Q for 24 h, then exposed to MNNG at various concentrations (10, 50, or 100 μM). Both NA + NM + R and NA + NM + Q combinational approaches showed significant enhancement of cell-protective effects compared to NA + NM treatment (Figure 1B). Though NA + NM pretreatment did not show a protective effect in cells exposed to MNNG at a high concentration (100 µM), minor cell-protective effects were observed in cells exposed to MNNG at lower concentrations (10 μM and 50 μM).

Q + NA + NM Approach Demonstrated the Highest Cell Rescue Effect in HEK293 Cells Under MNNG-Induced DNA Damaging Attack

To analyze the cell rescue effects of the phytocompounds and their combinations, HEK293 cells were first exposed to 100 μM MNNG treatment for 1 h, followed by treatments with individual compounds and their combinations with each other for 48 and 72 h. As shown in Figure 1C, NM, NA + NM, NM + Q, and NM + R treatments demonstrated significant cell rescue effects at both 48 and 72 h post-MNNG treatment, while NA, Q, R, NA + Q, NA + R, Q + R did not show any cell rescue effects when compared with the MNNG treatment control. These findings clearly revealed that NM was the essential agent responsible for rescuing HEK293 cells from MNNG triggered cell death and the NA + NM combination performed the best in this perspective. Based on these observations, we further investigated the cell rescue effects of the NA + NM + R and the NA + NM + Q combination approaches in comparison with the NA + NM approach in rescuing cells exposed to MNNG at a high concentration. As shown in Figure 1D, NA + NM + Q demonstrated the highest cell rescue effect, followed by NA + NM and then by NA + NM + R.

PARP1 Expression Levels Were Inversely Correlated With the Extent of MNNG-Induced DNA Damage and Positively Correlated to the Cell Rescue Effects Bestowed by the Combinational Approaches

In order to understand the underlying molecular mechanisms of these combinational approaches in affecting cell survival and DNA repair, we examined the expression levels of the DNA damage biomarker gamma-H2AX and the DNA damage repairing enzyme protein PARP1. As a well-established DNA damage indicator, gamma-H2AX is the phosphorylated form of H2AX, which is specifically phosphorylated by DNA damage-responding kinases such as ataxia-telangiectasia mutated and Rad3-related (ATR). ²⁷ In DNA repairing processes, PARP1 functions as the first responder enzyme that binds to damaged DNA and facilitates various DNA repairing activities, ²⁹ including single-strand DNA (ssDNA) break repairing. ³⁰ In the absence of PARP1, when ssDNA breaks are encountered during DNA replication, the replication fork stalls at the damaged DNA position, leading to the arrest of cell replication or even cell death. As demonstrated in Figure 2A and B, when cells were rescued from MNNG exposure at a lower concentration (10 μM), an inverse correlation was observed between the gamma-H2AX and the PARP1 expression levels, i.e., the sequential order (from high to low) of the gamma-H2AX levels was MNNG, NA + NM, NA + NM + Q and NA + NM + R, while it was NA + NM + R, NA + NM + Q, NA + NM and MNNG with the PARP1 levels (Figure 2A and B). The sequential order of the DNA damage extent was further confirmed by immunofluorescent imaging data (Figure 3). Additionally, the results also demonstrated that though the NA + NM approach already significantly increased the DNA repair activity by upregulating PARP1 levels, the addition of quercetin and resveratrol further potentiated the DNA repair activity of NA + NM.

Figure 2.

Detection and quantification of gamma-H2AX and PARP1 levels in MNNG-treated HEK293 cells, followed by rescuing cells with NA + NM, NA + NM + Q and NA + NM + R. (A) HEK293 cells were pretreated with MNNG (10 μM) and DMEM medium alone for 1 h, followed by media exchange with complete DMEM, NA + NM, NA + NM + Q or NA + NM + R and continuously incubated for 48 h. The gamma-H2AX and PARP1 levels were detected by Western blotting. (B) Western blotting band intensities in (A) were expressed as relative expression levels to their beta-actin loading controls using Image J software. Data show the mean ± SD (three independent experiments). ^∗P < .05; ^∗∗P < .01; ^∗∗∗P < .001.

Figure 3.

Detection of DNA broken foci by gamma-H2AX immunofluorescent staining to show DNA damage extent in MNNG-exposed HEK293 cells followed by rescue treatments. HEK293 cells were pretreated with MNNG (10 μM) for 1 h, followed by NA + NM, NA + NM + Q, and NA + NM + R treatments for 48 h. DNA broken foci formation was detected using anti-gamma-H2AX antibody. Green color indicates Gamma-H2AX expression while blue color indicates DAPI stained nuclei. The images were taken by a fluorescence microscope. Scale bar = 20 μm.

NA + NM Combination Approach Showed a Comparable NAD⁺ Boosting Effect as That of the NMN Approach

It has been reported that NA and NM have additive effects on raising NAD⁺ levels in human cells. ³¹ However, a side-by-side comparison of this combinational effect in boosting NAD⁺ levels with other NAD⁺ precursors has not been reported previously. In this study, we compared the in vitro NAD⁺ boosting effect of the NA + NM approach with NMN, a powerful NAD⁺ boosting precursor which has recently drawn much research and commercial attention. As shown in Figure 4, the absolute NAD⁺ level in NA + NM treated HEK293 cells were raised from 26.74 ± 6.53 nM to 69.11 ± 4.03 nM, and the NAD⁺/NADH ratio was raised from 0.75 ± 0.25 to 2.25 ± 0.30. In comparison, the raised absolute NAD⁺ level was 76.47 ± 7.34 nM and the raised NAD⁺/NADH ratio was 2.37 ± 0.52 in NMN-treated cells. These results clearly indicate that both NAD⁺ levels and NAD⁺/NADH ratios were significantly increased by both NA + NM and NMN approaches, and the NAD⁺ boosting effects of the two approaches were very comparable.

Figure 4.

Comparison of NAD⁺ boosting effect of NA + NM with NMN in HEK293 cells. (A) HEK293 cells were treated with complete DMEM, NA (5 mM) + NM (5 mM), and NMN (5 mM) for 24 h, followed by washing with ice-cold PBS. Total NAD and NADH amounts were measured using NAD⁺/NADH Quantification Colorimetric Kit. The absolute NAD⁺ value was calculated by subtracting the NADH value from the total NAD value (NADH and NAD⁺). (B) NAD⁺/NADH ratio was calculated by dividing the NAD value by the NADH value. Data are shown as mean ± SD. ∗, P < .05; ∗∗, P < .01.

Discussion

It is estimated that approximately 70,000 DNA lesions are generated daily in each human cell. ³² The majority of these (75%) are ssDNA breaks which are mainly caused by excessive oxidative stresses arising by either cell metabolic procedures or DNA base hydrolysis. If left un-repaired, these ssDNA breaks can progress to double-strand DSBs, which are more deleterious and can lead to many human diseases. For example, if the unrepaired DNA damage lesions accumulate in non-replicating cells, such as brain or muscle cells of adult mammals, these cells can become a major cause of organ aging. ³³ If the unrepaired DNA lesions pass on in replicating or stem cells, such as epithelium lining or bone marrow cells, the inherited DNA errors can give rise to gene mutations or epigenetic alterations, and both could become a major cause of tumorigenesis. ³⁴ Given the extraordinarily high frequency of DNA damaging events and the deleterious consequences of un-repaired DNA errors, there has been increasing interest in developing efficient natural products that can either prevent cells from DNA damaging or facilitate repairing DNA lesions to restore cells’ genome stability.

Owing to its dual activities in facilitating DNA repair and in consuming the intracellular NAD⁺, PARP1 plays a pivotal role in determining the cell's fate in dealing with DNA damaging stresses. ³⁴ On the one hand, PARP1 facilitates multiple DNA repair mechanisms and, therefore, plays a key role in rescuing cells from DNA damage-triggered apoptotic cell death. On the other hand, if too much PARP1 is activated, it can cause significant consumption of NAD⁺ and an energy crisis, leading to the pathway of necrotic cell death.^35,36 Therefore, it is plausible to speculate that an ideal intervention approach for managing DNA damaging stresses should fulfill the following two aspects: (i). It should maintain the intracellular NAD⁺ pool to a relatively high level to prevent cells from ATP depletion-induced necrotic cell death; (ii). It should maintain PARP1's activity to an optimal level to protect cells from DNA damage-induced apoptotic cell death.

In the present study, we first identified that both quercetin and resveratrol can significantly protect HEK293 cells from a DNA-damaging agent, MNNG, induced cell death. Given the findings that the NA + NM combination approach did not show a detectable cell protection effect in comparison with the control (Figure 1B), and that NA + NM co-treatment leads to a significant increment of intracellular NAD⁺ (Figure 4), it is plausible to speculate that the cell protection effect bestowed by NA + NM + Q and NA + NM + R was not due to increased NAD⁺ levels, but possibly due to the increased DNA repairing activity attributed to enhanced PARP1 expression, as indirectly supported by the findings derived from the cell rescue experiment (Figure 2).

However, we reasoned that increased intracellular levels of NAD⁺ were more likely responsible for the observed cell rescue effects in NM, NM + NA, NM + R, NM + Q, NA + NM + R, and NA + NM + Q treated cells (Figure 1C and D). This is because a cell rescue effect was observed in all the treatments that involved NM; and that NM is a well-known and highly efficient NAD⁺ precursor in boosting NAD⁺ levels. Interestingly, though NA significantly potentiated the cell rescue effect of NM, NA alone did not show any cell rescue effect in the study. The reason may be that NA cannot quickly boost NAD⁺ level as much as NM does.

In comparison with the NA + NM approach in the cell rescue experiment (cells were pretreated with MNNG at a high concentration), the NA + NM + Q combination demonstrated an increase, while NA + NM + R demonstrated a decreased cell rescue effect (Figure 1D). We speculated that this was because of quercetin's potent CD38 inhibitory and mild PARP1 inhibitory activities,^26,37 as inhibition of either CD38 or PARP1 would significantly preserve the intracellular pool of NAD⁺ and, therefore, rescue more cells from NAD⁺ depletion-caused necrotic cell death. As for the NA + NM + R combinational approach, since resveratrol was recently revealed to be a PARP1 activator via the distinctive tyrosyl-tRNA synthetase-dependent mechanism,^25,38 it is not difficult to understand a decreased cell rescue effect with the NA + NM + R approach, attributed to the NAD⁺ consuming activity of PARP1. It is worthwhile to point out that though PARP1 activation caused less cell rescue from NAD⁺ depletion-triggered necrotic cell death, it does enhance the cell's genome stability and therefore helps by protecting cells from more DNA-damaging attack, which was evidenced by decreased Gamma-H2AX expression (Figures 2, 3), and more protection of cells from DNA damaging attack-induced cell death (Figure 1B).

The NAD⁺ boosting effects were also compared between the NA + NM and NMN approaches. Comparable effects were detected between the two approaches in boosting the absolute NAD⁺ value and the ratio of NAD⁺/NADH (Figure 4). This finding provided more scientific evidence for guiding new formulation developments of NAD⁺ boosting natural products in the future.

Conclusion

The present study demonstrated that the NA + NM + R and NA + NM + Q combination approaches significantly improved the cell protection and rescue effects rendered by NAD⁺ precursors, NA and NM. The underlying mechanisms are plausibly related to increased intracellular NAD⁺ levels and elevated PARP1 activity.