Porcine Placental Extract Increase The Cellular NAD Levels in Human Epidermal Keratinocytes Ⅱ

Discussion 

Many studies have shown that the decrease in NAD due to aging is correlated with the functional decline of tissues and the development of age-related diseases and cancer11,22. In the epidermis, UV irradiation, the most important extrinsic factor of skin aging, causes NAD depletion as well as chronological aging23. Since the skin is constantly exposed to UV irradiation in daily life, the topical application of NAD-boosting agents seems to be a reasonable approach for preventing NAD reduction and protecting the skin from UV damage. Indeed, enhancing NAD in the skin has been shown to prevent UV-induced skin cancer and immunosuppression in mice24. Jacobson et al. reported that topical nicotinic acid derivatives increased skin NAD content and improved barrier function25.

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 In addition, several clinical studies have shown that topical application of the NAD precursor NAM reduces signs of skin aging26,27. These in vivo and clinical data support the possibility that elevated NAD levels prevent skin damage and delay aging. We have also previously reported that maintaining adequate NAD levels is essential for the protection of human epidermal cells from UV stress21. In this study, we demonstrated that PPE enhances intracellular NAD levels in keratinocytes. We also found that its active ingredients have LMW of less than 3 kDa, which is efective for both monolayer and 3-dimensional epidermis culture models. These findings suggest that the active ingredients of PPE could be transdermally absorbed and therefore highlight its potential application as a topical agent for boosting skin NAD levels.

Table 1. NAD precursor contents of PPE.

In the skin, NAD deficiency lowers the function of the main energy metabolic pathways, such as glycolysis and oxidative phosphorylation. Glycolysis is indispensable for maintaining a balance between keratinocyte proliferation and differentiation28. UV exposure decreases NAD levels and therefore may accelerate this imbalance. The ratio of differentiation to proliferation was observed to be profoundly increased in sun-exposed skin compared to sun-protected skin29. Tan et al. found that FK866 treatment accelerated differentiation and decreased metabolic activity in NHEKs, and that NAM reversed these FK866-induced events by restoring cellular NAD levels28. 

Figure 4. Topical PPE shows NAD recovery activity but does not affect cell metabolic activity, in RHE. (A) Schematic illustration of the RHE model. (B) PPE (2 mg/mL) was applied to the stratum corneum side of RHE and incubated for 48 h in the presence or absence of FK866 (5 nM). The total NAD levels of tissues were determined using an NAD+/NADH assay kit. (C) PPE (0, 1, 2, or 10 mg/mL) or 1% TX-100 (as the positive control) was applied to the stratum corneum side of RHE and incubated for 48 h. The cell metabolic activity was determined by MTT assay. **p<0.01, the comparison to the control.

In the present study, we found that PPE restored the FK866-induced NAD depletion. Therefore, determining whether PPE can sustain differentiation and proliferation in the NAD-depleted epidermis may provide a better understanding of its anti-aging mechanism.

Recently, Aioi et al. showed that topical use of PPE greatly reduced facial wrinkles and improved skin hydration17. Our previous clinical test showed that topical application of PPE cream resulted in a significant improvement in anti-skin aging parameters, such as wrinkles, skin hydration, sagging, and eyebags (unpublished data). The findings of this study may contribute to the understanding of the underlying mechanism by which PPE reduces wrinkles and improves the hydration of the skin. A previous report showed that intracellular total NAD levels are positively correlated with the expression of extracellular matrix components, such as procollagen and elastin, in human skin fbroblasts30. The treatment of fibroblasts with PPE results in increased production of type I collagen17. Combined with these facts, PPE may promote collagen production by increasing NAD content within fibroblasts, which may lead to the alleviation of skin wrinkles observed in clinical trials. Skin hydration is closely associated with the barrier function of the epidermal layer. Several studies have suggested that NAD is an essential co-factor for maintaining skin barrier function. Ming et al. showed that the NAD-dependent deacetylase SIRT1 regulates the expression of filaggrin, which plays a critical role as a precursor protein of natural moisturizing factors in skin barrier integrity31. In addition, a clinical study demonstrated that the topical application of a NAD-boosting agent markedly decreased transepidermal water loss, together with an increase in stratum corneum thickness in photodamaged skin25. These reports suggest that PPE may enhance skin barrier function by increasing cellular NAD levels in the epidermis, probably leading to improved skin hydration noted in clinical trials.

In recent years, NAD boosters have been shown to be useful in enhancing antioxidant and anti-inflammatory effects. In diabetic and septic mouse models, NR administration significantly reduced high-fat diet-induced brain inflammation and prevented lipopolysaccharide-induced endothelial oxidative stress, respectively 32,33. Similar to NR, NMN supplementation in aged mice has been shown to reverse the pro-inflammatory mRNA expression profile in the neurovascular unit and to improve cardiovascular function by decreasing oxidative stress34,35. In addition, a clinical trial in aged men revealed that oral NR decreased the levels of circulating inflammatory cytokines36. Taken together, these in vivo and clinical studies support the idea that the application of NAD-boosting agents provides antioxidant and anti-inflammatory effects. Enhanced SIRT activity has been suggested as one of the plausible mechanisms by which these NAD boosters exert anti-inflammatory and antioxidant effects. PPE is known to have antioxidant and anti-inflammatory effects15; however, relatively limited mechanistic exploration has been conducted to date. The present study provides at least a partial explanation of the effects of PPE.

PPE is known to contain abundant cell growth-promoting proteins such as laminin, cytokines, and growth factors. Recent studies, including ours, have implicated cellular NAD levels to be associated with cell growth in epidermal keratinocytes21,28. Hence, we speculated that some of these proteins might contribute to the increase in NAD content. However, contrary to our expectations, we found that a portion containing low-molecular-weight molecules of less than 3 kDa promoted NAD production in keratinocytes. Interestingly, fractionation of the PPE portion using HPLC revealed that NAD production was only observed in fraction 3. We also found that precursors of NAD, such as NMN, NR, and NAM, were present in this fraction, suggesting that they could explain the effect of PPE on NAD production, at least in part. However, since each constituent contained in PPE was detected at very low concentrations, it seems difficult to explain that these constituents alone are responsible for the ability of PPE to promote NAD content in keratinocytes. Recent reports have shown that the reduced forms of these NAD precursors, NRH and NMNH, have a greater ability to produce NAD than NR and NMN37–40. Although further analyses are needed to determine whether PPE contains NRH and NMNH, the NAD-boosting ability of PPE observed in this study may be attributed to multiple NAD enhancers, including NMN, NR, NMNH, NRH, and other unknown NAD precursors.

Materials and methods

temperature in a desiccator, the weight was measured with a precision balance. Te dry solid content of PPE was 10.98 mg/mL.

Cell cultures. 

NHEKs (newborn/male, Termo Fisher Scientifc, Waltham, MA, USA) were cultured in EpiLife™ medium containing 60 μM calcium supplemented with human keratinocyte growth supplement (both from Termo Fisher Scientifc) at 37 °C in a humidified atmosphere with 5% CO2. Te medium was changed every alternate day until the culture reached approximately 50% confluence, after which it was changed every day. The cells were passaged when they reached 80–90% confluency. NHEKs were not used beyond passage 5.

NAD quantification and recovery assay. 

The amount of NAD was measured using the NAD+/NADH Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 1.0× 105 NHEKs were seeded into 12-well plates. After 24 h, cells were treated with PPE at different concentrate options (0, 0.25, 0.5, and 1 mg/mL) and cultured for 1–24 h in the presence or absence of 5 nM FK866 (AdooQ Bioscience, Irvine, CA, USA). The cells were lysed with the supplied NAD+/NADH extraction buffer and ultra-filtered using an Ultracel-10 K centrifugal filter device (Merck, Darmstadt, Germany). To calculate NADH concentration, half of the filtrates were incubated at 60 °C for 60 min to decompose NAD+. All filtrates were subjected to the enzymatic reaction at 37 °C for 1 h. Then, the absorbance of the samples was measured at 450 nm using an Infinite 200 Pro plate reader (Tecan, Männedorf, Switzerland). The NAD+ content was calculated by subtracting the NADH content from the total NAD content. NAD+/NADH levels were normalized to the respective protein concentrations determined using the Pierce BCA protein assay (Thermo Fisher Scientific).

with 300 μL of 1 mg/mL MTT (Thermo Fisher Scientific) solution at 37 °C and 5% CO2 for 3 h. After washing three times with PBS, formazan infiltrated in NHEKs was extracted with 200 μL of dimethyl sulfoxide at room temperature for 2 h. Optical absorption at 560 nm was determined using an Infnite 200 Pro plate reader (Tecan).

Ultrafiltration of PPE. PPE (500 μL) was applied to an Ultracel-3K centrifugal flter device (Merck Millipore) and centrifuged at 16,100×g at 4 °C for 60 min. The filtrates were used as an LMW fraction of PPE. To recover the residues on the filter, the filter devices were placed upside down in a new centrifuge tube and centrifuged at 1000×g at 4 °C for 5 min. Te residues were reconstituted with 500 μL of phosphate-buffered saline (PBS) and used as an HMW fraction of PPE.

min: hold at 100% solvent A from 0 to 10 min, then a linear gradient from 0% B in 10 min to 95% B in 30 min. Te eluents were collected in sample tubes every 2 min and dried using a centrifugal evaporator CVE-3100 (EYELA, Tokyo, Japan). The residues were reconstituted with 100 μL of PBS and filtered through a 0.22 μm membrane before using the NAD recovery assay.

Analyses of NAD precursors. 

NMN, NR, and NAM were detected and quantified by isotope dilution LC–MS/MS. Analyses were performed using an ACQUITY UPLC H-Class system coupled with a TQS-micro triple quadrupole mass spectrometer (Waters, Milford, MA, USA) with electrospray ionization operated in positive ion mode. A Capcell Pak ADME HR column (2 μm, 2.1×100 mm, Osaka Soda, Osaka, Japan) was used to separate each sample. Mobile phases A–B were water–methanol (containing 0.1% formic acid and 10 mM ammonium formate). The gradient condition was as follows: 0–0.5 min (99% A), 0.5–3 min (99–60% A), 3–5 min (60–2% A), and 5–7 min (2% A). Te flow rate was 0.2 mL/min. The capillary voltage was 0.75 kV, the source temperature was 150 °C, and the desolvation temperature was 500 °C. The cone gas flow was 50 L/h and the desolvation gas flow was 1100 L/h. To quantify each NAD precursor, NAM-13C6 was added to the samples at a concentration of 78 nM as an internal standard. The ions were monitored using multiple reaction monitoring (MRM) with the following transitions: m/z 335→123 for NMN, m/z 255→123 for NR, m/z 123→80 for NAM, and m/z 129→85 for NAM-13C6. NMN was purchased from the Oriental Yeast (Tokyo, Japan). NR and NAM-13C6 were obtained from Merck (Darmstadt, Germany). NAM was purchased from Wako Pure Chemical Industries (Osaka, Japan).

RHE. The in vitro RHE model EpiDerm™ (EPI-200) was purchased from MatTek (Ashland, MA, USA) and cultured in EPI-100-ASY medium (MatTek) at 37 °C and 5% CO2. One hundred microliters of PPE (1, 2, and 10 mg/mL) or 1% TX-100 was applied to the stratum corneum side of the RHE. The RHE was incubated for 48 h in an EPI-100-ASY medium in the presence or absence of FK866 (5 nM). After incubation, the RHE was washed three times with PBS. For the NAD recovery assay, the total NAD levels in RHE were quantified using the NAD+/NADH Assay Kit-WST. For the cell metabolic activity assay, the RHE was transferred to a 24-well plate and incubated with 300 μL of 1 mg/mL MTT (Thermo Fisher Scientific) solution at 37 °C and 5% CO2 for 3 h. After washing three times with PBS, formazan infiltrated in RHE was extracted with 2 mL of dimethyl sulfoxide at room temperature for 2 h. Optical absorption at 560 nm was determined using an Infnite 200 Pro plate reader (Tecan). The absorbance with 1% TX-100 treated RHE was used as the positive control for the cell metabolic activity assay. 

Statistical analysis. Data are presented as the mean±standard error of the mean of at least three independent experiments. All statistical analyses were performed using the EZR software version 1.55 (Saitama Medical

References

Appl. 11, 101–109 (2021).

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