ATP supplementation suppresses UVB-induced photoaging in HaCaT cells via upregulation of expression of SIRT3 and SOD2.

Background: Skin photoaging is the damage caused by excessive exposure to ultraviolet (UV) irradiation. We investigated the effect of adenosine triphosphate (ATP) supplementation on UVB‐induced photoaging in HaCaT cells and its potential molecular mechanism. Materials and methods: The toxicity of ATP on HaCaT cells was examined by the MTT assay. The effects of ATP supplementation on the viability and apoptosis of HaCaT cells were determined by crystal‐violet staining and flow cytometry, respectively. Cellular and mitochondrial ROS were stained using fluorescent dyes. Expression of Bax, B‐cell lymphoma (Bcl)‐2, sirtuin (SIRT)3, and superoxide dismutase (SOD)2 was measured via western blotting. Results: ATP (1, 2 mM) exerted no toxic effect on the normal growth of HaCaT cells. UVB irradiation caused the apoptosis of HaCaT cells, and ATP supplementation inhibited the apoptosis induced by UVB significantly, as verified by expression of Bax and Bcl‐2. UVB exposure resulted in accumulation of cellular and mitochondrial reactive oxygen species (ROS), but ATP supplementation suppressed these increases. Expression of SIRT3 and SOD2 was decreased upon exposure to UVB irradiation but, under ATP supplementation, expression of SIRT3 and SOD2 was reversed, which was consistent with the reduction in ROS level observed in ATP‐treated HaCaT cells after exposure to UVB irradiation. Conclusions: ATP supplementation can suppress UVB irradiation‐induced photoaging in HaCaT cells via upregulation of expression of SIRT3 and SOD2.

Keywords: ATP; ROS; SIRT3; skin photoaging; SOD2

Excessive ultraviolet (UV) radiation is the main factor influencing skin photoaging. It has been reported that 90% of skin diseases are related to UV irradiation.[1] UVA (320–400 nm) and UVB (280–320 nm) account for >90% of solar radiation, the other is UVC (200–280 nm).[1] UVA can penetrate and reach the dermis of the skin directly to destroy elastic fibers and collagen fibers, thereby accelerating the formation of melanin and darkening the skin tone.[[2], [4]] UVB can cause epidermal swelling and sunburn in a short time.[[5]] Hence, long‐term exposure to UVA or UVB can cause skin damage (and even skin cancer).

Reactive oxygen species (ROS) are formed as natural byproducts of normal aerobic metabolism. They have important roles in cell signaling and homeostasis.[[7]] ROS are intrinsic to cellular functioning and present at high levels under the stimulation of UV irradiation. ROS beyond the antioxidant capacity of cells can induce severe oxidative damage, including apoptosis, aging, and even cancer. Excessive ROS can not only result in peroxidation of macromolecular substances in cells to induce oxidative damage[[9]] but also activate signaling pathways connected with apoptosis to promote programmed cell death.[11] Even though the skin has effective mechanisms to defend against oxidative stress, ROS overload has a crucial influence upon skin photoaging.[12]

There is considerable evidence that various enzymatic and nonenzymatic antioxidants can decrease ROS accumulation to further reduce ROS‐mediated apoptosis and oxidative damage.[8] Superoxide dismutase (SOD)2 is an antioxidant metal enzyme that plays a key part in redox balance. Several studies have found that mitochondrial ROS can be eliminated by SOD2, and that its activity is associated with sirtuin (SIRT)3.[[13]] The latter is a nicotinamide adenine dinucleotide‐dependent deacetylase localized in mitochondria, and regulates oxidative stress.[15] Abnormal expression of SIRT3 affects mitochondrial function negatively. Some studies have suggested that SIRT3 overexpression could suppress oxidative stress‐mediated neurotoxicity and mitochondrial dysfunction in dopaminergic neuronal cells.[16]

Adenosine triphosphate (ATP) is found in all known forms of life. It provides energy to drive many processes in living cells and maintain intracellular homeostasis.[[17]] Imbalance of ROS levels would induce mitochondrial dysfunction, which in turn results in depolarization of the mitochondrial membrane potential. Complexes I–IV of the mitochondrial respiratory chain and ATP content are also altered with mitochondrial dysfunction.[[19]] Some reports have suggested H2O2‐induced mitochondrial dysfunction to be related to a decrease in the ATP level.[[21]]

Here, we investigated the special effect of ATP supplementation on UVB‐induced photoaging in HaCaT cells and the potential mechanism of action.

Adenosine 5′‐triphosphate disodium salt hydrate (ATP) was purchased from Aladdin (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco (Grand Island, NY, USA). Fetal bovine serum was sourced from Biological Industries (Beit HaEmek, Israel). Penicillin/streptomycin solution, radioimmunoprecipitation (RIPA) lysis buffer, and phenylmethylsulfonyl fluoride were from Solarbio (Beijing, China). Phenol red‐free medium was purchased from Thermo Fisher Scientific (Waltham, MA, USA). An annexin V‐fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit was sourced from Beijing 4A Biotech (Beijing, China). Antibodies against SIRT3, SOD2, B‐cell lymphoma (Bcl)‐2, and Bax were obtained from Cell Signaling Technology (Beverly, MA, USA). The antibody against β‐tubulin was sourced from Sino Biological (Beijing, China).

Human HaCaT cells were purchased from Conservation Genetics CAS Kunming Cell Bank (Kunming, China). HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution in a humidified incubator in an atmosphere of 5% CO2 at 37°C.

The toxicity of ATP on cell growth was evaluated by the MTT assay. Briefly, HaCaT cells were plated and cultured in a 96‐well plate overnight. Then, cells were treated with ATP (0, 1, or 2 mM) in serum‐free medium for 24 h. Next, HaCaT cells were incubated with MTT solution for 4 h and dimethyl sulfoxide was added to the 96‐well plate. The optical density at 492 nm was measured through a microplate reader (FlexStation 3; Molecular Devices, Sunnyvale, CA, USA). Percent cell viability was calculated and compared with that in the solvent control.

Viable HaCaT cells were cultured in 6‐cm culture dishes at 1 × 106 cells and incubated overnight. When they reached 50% confluence, cells were pretreated with ATP (1 or 2 mM) for 1 h in serum‐free medium, irradiated with a UVB lamp for 5 min, and incubated for 24 h. Subsequently, HaCaT cells were fixed in 4% paraformaldehyde for 20 min and then stained by a solution of crystal violet for 10 min. After washing with 1× phosphate‐buffered saline, the corresponding samples were imaged and recorded.

The apoptosis of HaCaT cells was determined using flow cytometry with a FACSCalibur system (BD Biosciences, San Jose, CA, USA) and the annexin V‐FITC/PI kit. HaCaT cells were exposed to ATP (1 or 2 mM) for 1 h and then treated with UVB light for 5 min. After incubation for 24 h, HaCaT cells in the medium and the culture dish were collected. After washing and resuspension, HaCaT cells were incubated with annexin V‐FITC (20 μL) for 20 min. After staining with PI (20 μL) for 15 min in the dark at room temperature, cells were filtered into a flow tube and analyzed by flow cytometry.

Intracellular and mitochondrial ROS were detected by 5‐(6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate, acetyl ester (CM‐H2DCFDA), and MitoSOX dye (Thermo Fisher Scientific, Waltham, MA, USA), respectively. HaCaT cells were seeded on a cell slide in a 12‐well plate and incubated overnight. When they reached 50% confluence, the culture medium was replaced with ATP (1 or 2 mM) in serum‐free supplement for 1 h then irradiated with a UVB lamp for 5 min. After incubation for 24 h, cells were stained by dye (5 mM) in phenol red‐free medium (1 mL) for 15 min. Images were acquired at 200× magnification under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). CM‐H2DCFDA is freely permeant to cell membranes and is oxidized to green‐fluorescent calcein by peroxides in the cytoplasm.[29] MitoSOX penetrates into living cells and targets mitochondria selectively, which can be oxidized rapidly by superoxide to produce considerable red fluorescence.[24] Intracellular ROS appeared as green fluorescence and mitochondrial ROS were visible as red fluorescence under a microscope. Images were analyzed through ImageJ (US National Institutes of Health, Bethesda, MD, USA).

HaCaT cells were lysed with RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride for 30 min on ice. The supernatant was collected after centrifugation (15 000×g, 10 min, room temperature). The protein concentration was determined by an Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins (20 μg) from each group were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 12% gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Finally, the PVDF membranes were incubated overnight at 4°C with the following primary antibodies (1:1000 dilution): anti‐Bcl‐2, anti‐Bax, anti‐SIRT3, anti‐SOD2, and anti‐β‐tubulin. PVDF membranes were treated with immunoglobulin‐g–horseradish peroxidase. Bands were visualized by enhanced chemiluminescence.

Prism 5.0 (GraphPad, La Jolla, CA, USA) was used for statistical analyses. The Student's t‐test was employed to compare data with a normal distribution between two groups. Results are the mean ± SEM. p < 0.05 was considered significant.

The MTT assay showed that ATP treatment did not affect the normal growth of HaCaT cells (Figure 1A). Studies have demonstrated the important role of ATP in the recovery of cell viability.[[19]] We detected the survival of UVB‐irradiated HaCaT cells under ATP intervention by crystal‐violet staining (Figure 1B). Significantly more viable HaCaT cells were observed in the control group than in the group with UVB‐irradiated HaCaT cells. However, increased viability of HaCaT cells was observed under ATP supplementation after UVB stimulation. These results suggested that ATP supplementation could protect HaCaT cells from the harmful effects of UVB irradiation.

Our previous experiment showed that ATP supplementation improved the survival of UVB‐irradiated HaCaT cells significantly. We detected the apoptosis of HaCaT cells with different treatments using flow cytometry. UVB irradiation increased the percent apoptosis of HaCaT cells significantly compared with that in the control group (Figure 2A). However, reduced percent apoptosis of HaCaT cells was shown upon ATP supplementation (Figure 2B). These results demonstrated increased apoptosis of HaCaT cells upon UVB irradiation, and that ATP supplementation could decrease the apoptosis of HaCaT cells caused by UVB irradiation.

Studies have shown that UV irradiation can induce ROS accumulation, which can activate apoptosis‐associated signaling pathways.[[25]] We found that apoptosis was increased after UVB irradiation, which led us to speculate on the connection between UVB irradiation and ROS accumulation. Staining and a fluorescent probe were used to reveal ROS accumulation in the cytoplasm and mitochondria. The ROS level in the cell (Figure 3A) and mitochondria (Figure 3C) in UVB‐induced HaCaT cells was significantly higher than that in the control group. The mean fluorescence intensity of cellular ROS (Figure 3B) or mitochondrial ROS (Figure 3D) was reduced markedly upon ATP treatment. These results demonstrated that UVB irradiation promoted the intracellular and mitochondrial accumulation of ROS, and that ATP supplementation inhibited the ROS content effectively.

We wished to determine the reason behind the reduction in the apoptosis of HaCaT cells in the ATP‐supplementation group. We measured the levels of Bax and Bcl‐2, the core proteins in apoptosis, in different groups using western blotting. Immunoblotting showed that expression of the proapoptotic Bax protein was increased significantly in UVB‐treated HaCaT cells (Figure 4A), whereas expression of the antiapoptotic Bcl‐2 protein was inhibited (Figure 4B). However, upon ATP supplementation, Bax expression was reduced markedly, and expression of Bcl‐2 protein was obviously upregulated, compared with those in UVB‐irradiated HaCaT cells. These data were consistent with our results from flow cytometry.

To further explore how ATP inhibits ROS accumulation in UVB‐irradiated HaCaT cells, expression of SIRT3 and SOD2 was measured by western blotting. Expression of SIRT3 (Figure 4C) and SOD2 (Figure 4D) was decreased markedly in UVB‐treated HaCaT cells compared with that in the control group. However, ATP supplementation recovered the expression of SIRT3 and SOD2 in UVB‐irradiated HaCaT cells. Changes in protein expression of SIRT3 and SOD2 were associated with reductions in ROS levels in UVB‐induced HaCaT cells upon ATP treatment. Collectively, these findings indicated that ATP supplementation could elicit a protective effect upon UVB‐irradiated HaCaT cells by modulating the SIRT3/SOD2 signaling pathway.

The skin is the largest organ in the human body. It serves as the first barrier to prevent external damage.[[27]] The skin is prone to various types of stimulation.[29] UV irradiation is the main factor causing skin photoaging and leads to numerous pathologic manifestations in the skin.[30] Usually, UVA induces damage to the dermis and UVB harms the epidermis of the skin.[[31]] Studies have reported that UVB induces potent ROS accumulation, resulting in oxidative damage to the skin.[33] Excessive ROS destroy the redox balance and shift it toward a pro‐oxidative state, thereby resulting in oxidative stress.[33] The detrimental effects of oxidative stress occur through multiple mechanisms that involve peroxidation to proteins and lipids, alterations of signaling pathways, and the induction of inflammation. Inhibition of oxidative stress is considered to be a rational approach to prevent skin photoaging.[34] Several components of the skin have been found to exhibit potent antioxidant capacity and the ability to counteract UV‐induced insults to the skin.[[35], [37]]

ATP is the "energy currency" in cells. It provides energy for activities in the body.[38] The body uses glucose to store energy in ATP through glycolysis and then releases energy through the tricarboxylic‐acid cycle and oxidative phosphorylation for muscle contraction, signal transmission, breathing, and other activities.[[39]] A considerable volume of experimental evidence suggests that excessive ROS beyond the antioxidant capability is related to a reduction in the ATP level in cells.[[39]] In the induction of UV‐mediated oxidative stress, ROS overload would alter the mitochondrial membrane potential, thereby interfering with ATP production.[[41]] Several studies have demonstrated that ATP supplementation can control energy metabolism, signal transmission, and the viability of cells.[43] We wondered whether ATP supplementation could protect the skin from UV irradiation by regulating an intrinsic molecular mechanism.

It is well known that UV irradiation can decrease cell viability and promote apoptosis. Therefore, we investigated the viability of HaCaT cells with or without ATP supplementation after UVB irradiation by crystal‐violet staining. The UVB‐irradiation model in human HaCaT cells was established in our study as described previously.[44] We discovered that UVB irradiation inhibited the proliferation of HaCaT cells. However, ATP supplementation increased the proliferation in HaCaT cells significantly after UVB irradiation. Besides, we showed that if HaCaT cells were exposed to UVB irradiation, percent apoptosis was increased significantly compared with that in the control group. As determined by flow cytometry, UVB irradiation increased percent apoptosis of HaCaT cells significantly compared with that in the control group. Under ATP supplementation, percent apoptosis was reduced in UVB‐irradiated HaCaT cells. These results demonstrated that ATP supplementation inhibited the apoptosis of HaCaT cells caused by UVB irradiation, which suggested the protective effect of ATP in HaCaT cells against the detrimental influences of UVB. Furthermore, UVB irradiation significantly promoted Bax expression and inhibited Bcl‐2 expression. Bax and Bcl‐2 are regarded as the regulators of apoptosis. However, Bax expression was inhibited markedly, and Bcl‐2 expression was increased significantly, in UVB‐irradiated HaCaT cells upon ATP supplementation. These results showed that ATP supplementation could suppress UVB‐induced skin photoaging by reducing apoptosis.

Excessive exposure to UV irradiation increases the production of free radicals in HaCaT cells and results in ROS accumulation, which in turn generates oxidative damage to the skin.[45] Therefore, we measured the ROS accumulation caused by UVB irradiation using a fluorescent probe. Cellular and mitochondrial levels of ROS were increased obviously in UVB‐treated HaCaT cells. However, ATP supplementation weakened the accumulation of intracellular and mitochondrial ROS in HaCaT cells after UVB irradiation. Due to a decrease in antioxidant activity caused by UV irradiation, ROS accumulation is increased and results in cell damage. SOD2 is a key regulator that can eliminate excessive ROS. Therefore, we considered whether ATP supplementation influences expression of SOD2 and its regulatory factor SIRT3 to inhibit ROS accumulation in UVB‐exposed HaCaT cells. UVB irradiation alone partially reduced the expression of SOD2 and SIRT3 compared with that in the control group, but ATP supplementation increased their expression markedly, in UVB‐irradiated HaCaT cells. These results indicated that the SIRT3/SOD2 pathway may be an important factor for ATP to exhibit a positive effect, but further studies on the mechanism of action are needed.

ATP supplementation could: (i) suppress UVB‐induced damage to HaCaT cells; (ii) reduce UVB‐induced apoptosis to HaCaT cells, along with downregulating protein expression of Bax and upregulating protein expression of Bcl‐2; (iii) decrease the accumulation of intracellular and mitochondrial ROS induced by UVB irradiation. Recovery of the protein expression of SIRT3 and SOD2 under ATP supplementation suggested that SIRT3/SOD2 may be the potential pathway for ATP to work on. ATP supplementation could become a novel strategy to inhibit UVB‐induced photoaging in HaCaT cells via upregulation of the SIRT3/SOD2 pathway.

This work was supported by grants from the Yunnan Fundamental Research Project (202101BD070001‐034, 202101AU070216, 202101BD070001‐049, 202101AU070086, and 202101AT070749), the Scientific Research Fund Project of Yunnan Provincial Education Office (2022J00297), and the Yunnan Provincial Key Programs of Yunnan Eco‐friendly Food International Cooperation Research Center Project (2019ZG00904 and 2019ZG00909).

The authors declare that they have no conflict of interests.

The data that support the findings of this study are available from the corresponding authors upon reasonable request.