Apigenin reduces oxidative damage in pigment cells by activating protective cell pathways and lowering harmful molecules in zebrafish

Oxidative damage models are commonly induced by H₂O₂. We first performed in vitro experiments. As shown in Figure 1A, apigenin significantly reduced the elevation of ROS caused by oxidative stress in zebrafish. In order to determine the effects of different concentrations of H₂O₂ on cell viability and intracellular ROS production, B16F10 cells were treated with various concentrations of H₂O₂ (0~1 mM) for a duration of 12 h. The effect of H₂O₂ on the cell viability was detected by MTT assay. As shown in Figure 1B, there was less cytotoxicity in the concentration range of H₂O₂ at 0.1~0.4 mM, which was selected for further experiments. And then, we examined the effect of H₂O₂ on ROS generation in B16F10 cells with the ROS assay. The results showed that 0.4 mM H₂O₂ caused less cellular damage and significantly increased the intracellular ROS levels (Figure S1B); therefore, 0.4 mM H₂O₂ for 12 h was finally chosen to fabricate the oxidative stress model of B16F10 cells.

The intracellular scavenging mechanisms of free radicals include two main types of antioxidant enzymes and small molecule antioxidants. Antioxidant enzymes include catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glyoxalase. As shown in Figure 1C,D, we found that apigenin could not only significantly reduce the accumulation of ROS in B16F10 cells, but also increase the activity of the antioxidant enzyme SOD and reduce the production of MDA (Figure 2A,B). As a conclusion, apigenin can attenuate oxidative damage caused by oxidative stress in B16F10 cells.

There is a significant link between Nrf2 and the pathogenesis of vitiligo. Nuclear transcription factors such as Nrf2 are essential for controlling oxidative stress in cells. When under oxidative stress, Nrf2 is translocated from the cytoplasm to the nucleus, where it binds to antioxidant response elements (AREs) located in the promoters of genes encoding antioxidant enzymes and controls the expression of related antioxidant enzymes (HO-1, NQO1, and SOD), thus exerting antioxidant effects. Therefore, we examined the effect of apigenin on the intracellular antioxidant Nrf2 signal pathway. The Western blot results showed that apigenin increased the expression of Nrf2, HO-1, and NQO1 proteins under oxidative stress (Figure 2C–G). Keap1, a part of the E3 ubiquitin ligase, ubiquitylates Nrf2, which is degraded by the proteasome, and the results indicated that apigenin can inhibit Keap1 expression and maintain Nrf2 stability. The above experimental results indicate that apigenin can activate the Nrf2/ARE signal pathway.

The β-galactosidase staining results showed that H₂O₂ significantly increased β-galactosidase activity in melanocytes, but apigenin pretreatment significantly inhibited the increase in the β-galactosidase activity induced by oxidative stress in the melanocytes (Figure 3A). The Western blot analysis was used to investigate the effects of apigenin on the levels of p53 and p21 proteins’ expression. The results demonstrated that cells treated with H₂O₂ showed an increase in the levels of p53 and p21 expression, while these changes were attenuated by apigenin (Figure 3B–D). This suggests that apigenin has the ability to attenuate the β-galactosidase activity and the p53 and p21 proteins’ expression in a dose-dependent manner, thus delaying cellular senescence.

To investigate the effects of apigenin on melanin synthesis affected by oxidative stress, we examined the levels of TYR, TRP1, TRP2, and gp100 proteins’ expression. As shown in Figure 4A–E, apigenin could increase the levels of TYR, TRP1, and TRP2 proteins’ expression in a dose-dependent manner. Furthermore, apigenin alleviated the inhibition of melanosome protein gp100 expression caused by oxidative stress. According to the findings, it is suggested that apigenin could regulate melanogenesis by increasing the inhibition of the expression of TYR, TRP1, TRP2, and gp100 induced by oxidative stress.

In order to examine whether apigenin can improve the atrophy of dendrites or pseudopods in oxidative damage melanocytes, we determined cytoskeletal changes by FITC–Phalloidin fluorescent probe staining and analyzed the expression of dendritic pseudopod regulation and adhesion-related proteins by Western blotting. In our investigation, it was observed that apigenin effectively enhanced the alleviation of dendrite atrophy induced by oxidative stress in melanocytes. Additionally, apigenin exhibited a significant influence on augmenting the elongation and quantity of F-actin (Figure 4F). In addition, Western blot results showed that 5 µM apigenin alleviated the suppression of Rac-1 and Cdc42 proteins’ expression caused by oxidative stress, and increased the expression level of E-Cadherin protein (Figure 4G–J). E-Cadherin is a class of membrane proteins that mediate cell adhesion and sensing of external information.

Mitochondria are organelles closely related to energy metabolism. Mitochondria can maintain the intracellular energy metabolism and homeostasis of the internal environment; once the mitochondria are damaged, the cell will lose vitality, and the mitochondrial membrane potential (MMP) that maintains the normal function of mitochondria plays an important role; it is an indispensable part of the process of cellular oxidative phosphorylation to generate energy. The decrease in MMP is also a sign of early apoptosis. When the MMP is higher, JC-1 aggregates and forms polymers in the mitochondrial matrix, resulting in red fluorescence; on the contrary, when the MMP is lower, JC-1 is unable to aggregate in the mitochondrial matrix and produces green fluorescence. The JC-1 staining results showed that the intensity of red fluorescence decreased, and green fluorescence increased significantly in B16F10 cells after H₂O₂ treatment compared with the control group, indicating that the mitochondria were damaged and dysfunctional. Compared with the model group, apigenin pretreatment effectively ameliorated the MMP decrease that was induced by oxidative stress (Figure 5A). The results indicated that apigenin protected the MMP, and enhanced the mitochondrial activity, which had a protective effect on the mitochondrial disorder in H₂O₂-treated B16F10 cells.

Mitochondrial fusion and fission are a dynamically balanced process within the cell and play a critical role in maintaining normal mitochondrial function. Therefore, we examined the key proteins’ expression during mitochondrial fusion and fission. As shown in Figure 5B–G, the expression of the fusion-associated protein (OPA1, Mitofusion-1) and the fission-associated protein p-DRP1(S637) were suppressed, and the p-DRP1(S616) and MFF proteins’ expression were increased in B16F10 cells under oxidative stress, indicating that mitochondrial fission was increased, and fusion was decreased. Apigenin could promote the expression of the above fusion proteins in B16F10 cells and p-DRP1(S637), and inhibit p-DRP1(S616) and MFF expression. These results suggest that apigenin promotes mitochondrial fusion, improves the imbalance between mitochondrial fusion and fission, improves mitochondrial membrane potential, and inhibits apoptosis in B16F10 cells under oxidative stress.

As shown in Figure 5H–N, the proteins of Beclin-1, Atg5, Atg8, Atg12, LC3-Ⅰ/II, and p62 expression were inhibited after H₂O₂ treatment in cells. Apigenin pretreatment, on the other hand, alleviated autophagy inhibition and dose-dependently promoted the levels of autophagy-related proteins’ expression. The results indicated that oxidative stress inhibits autophagy in B16F10 cells, and apigenin can reduce the oxidative damage of melanocytes under oxidative stress by promoting cellular autophagy, maintaining the normal state and function of cells, and reducing apoptosis.

Mitochondrial autophagy is the removal of senescent and damaged mitochondria by cells through selective autophagy. Therefore, we examined the levels of PINK1, Parkin, and BNIP3L/Nix proteins’ expression during mitochondrial autophagy. As shown in Figure 6A–D, after H₂O₂ treatment, the cells showed reductions in BNIP3L/Nix, PINK1, and Parkin expression. Apigenin (5 µM) pretreatment rescued the expression of BNIP3L/Nix, PINK1, and Parkin proteins after H₂O₂ treatment. These results suggested that apigenin might promote mitochondrial autophagy in B16F10 cells by activating PINK1/Parkin pathways.

The PI3K/Akt/mTOR pathway is known to be a pathway that regulates autophagy and is a negative regulator of autophagy. With activation of this pathway, cellular autophagy is inhibited. Therefore, we tested the effect of apigenin on the PI3K/Akt/mTOR pathway in B16F10 cells under oxidative stress. We detected PI3K, p-Akt, Akt, p-mTOR, and mTOR proteins’ expression by Western blot. The results showed that the above proteins’ expression was increased by oxidative stress, whereas apigenin or PI3K inhibitor (LY294002) inhibited the expression of the above proteins in the state of oxidative stress, and the cotreatment of both of them enhanced the inhibitory effect further. In addition, we found that apigenin and rapamycin (RAPA) inhibited mTOR phosphorylation and p62 up-regulation in B16F10 cells, and again, the inhibitory effect was enhanced by the combination of them. The above results suggest that apigenin inhibited the PI3K/Akt/mTOR pathway to reduce the promotion of ROS and then promoted autophagy in B16F10 cells under oxidative stress (Figure 6E–K).

Apigenin, obtained from Sigma-Aldrich Corporation (CAS: 10789-100MG, with purity ≥ 95.0%, St. Louis, Missouri, MO, USA), was prepared with dimethyl sulfoxide (DMSO) at the concentration of 100 mM. It was stored as small aliquots at −20 °C. Before adding to cell culture medium, they were melted and diluted to appropriate concentration. Antibiotic penicillin/streptomycin, trypsin, and enhanced chemiluminescence (ECL) solution were purchased from Biosharp (Hefei, China). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Carlsbad, CA, USA). The cell lysis buffer and PI3K inhibitors (LY294002) were purchased from Biyotime (Nanjing, China). H₂O₂ (323381-25ML, with purity 3 wt.% in H₂O, USA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against GAPDH (60004, 1:1000), β-actin (3700, 1:2500), TYR (ab180753, 1:2000), TRP1 (ab3312, 1:200), TRP2 (ab74073, 1:1000), and gp100 (ab137078, 1:1000) were purchased from Abcam (Cambridge, UK). Keap1 (8047, 1:1000), Nrf2 (12721, 1:1000), HO-1 (43966, 1:1000), NQO1 (62262, 1:1000), Rac-1 (4651, 1:1000), Cdc42 (2466, 1:1000), E-Cadherin (3195, 1:1000), p62 (39749, 1:1000), p53 (2524, 1:1000), p21 (37543, 1:1000), MFF (84580, 1:1000), p-DRP1(S616) (4494, 1:1000), p-DRP1(S637) (6319, 1:1000), DRP1 (8570, 1:1000), Mitofusion-1 (14139, 1:1000), OPA1 (80471, 1:1000), Beclin-1 (2495, 1:1000), Atg5 (12994, 1:1000), Atg8 (64459, 1:1000), Atg12 (4180, 1:1000), LC3Ⅰ/II (12741, 1:1000), PI3K (4263, 1:1000), p-Akt (4060, 1:2000), Akt (9272, 1:1000), p-mTOR (5536, 1:1000), mTOR (2983, 1:1000), BNIP3L/Nix (12396, 1:1000), PINK1 (6946, 1:1000), Parkin (4211, 1:1000), second antibody anti-mouse IgG (7076, 1:2500), and anti-rabbit IgG (7074, 1:2500) were obtained from Cell Signaling Technology (Danvers, MA, USA). The BCA protein assay kit (P009), reactive oxygen species assay kit (DCFH-DA, S0033S), cellular senescence-associated β-galactosidase staining kit (C0602), and mitochondrial membrane potential assay kit (JC-1, CA1310) were purchased from Biyotime Biotechnology (Nanjing, China). The FITC–Phalloidin (No. 40735ES80) was purchased from Shanghai Yeasen Biotechnology Co. Ltd. (Shanghai, China). The Superoxide Dismutase (SOD) assay kit (E-BC-K020-M), Catalase (CAT) activity assay kit (E-BC-K031-S), and Malondialdehyde (MDA) assay kit (E-EL-0060) were purchased from Elabscience (Wuhan, China).

B16F10 cells were purchased from the Cell Bank (the Chinese Academy of Science, China, Shanghai). These cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin, and placed in a 37 °C, 5% CO₂ incubator. An amount of 2.5% FBS was used when cells were treated with various concentrations of apigenin (0.01~10 μM) or under the condition of 48 h at 37 °C and with various concentrations of H₂O₂ (0.1~1 mM) to model oxidative stress for 12 h at 37 °C.

Adult zebrafish were obtained from Shanghai FishBio Co., Ltd. (China, Shanghai). Zebrafish were used to assess the depigmenting effects of pterostilbene in vivo with some modifications to a previous study [10]. Briefly, we kept the water temperature at about 28.5 °C, and they needed 14 h of light per day. Zebrafish embryos (collected after natural spawning was completed) were grouped into a 6-well plate by maintaining 30 embryos/well, and cultured in a light incubator at 28.5 °C. The embryos were treated or not with H₂O₂ (0.5 mM) for 4 h, in egg water containing apigenin (10 µM) for 24 h. The effects of apigenin on the ROS levels of zebrafish were observed using a Leica M205 FCA stereomicroscope (Germany, Heidelberg).

The viability of B16F10 cells was determined by the MTT kit was purchased from Biyotime Biotechnology (China, Nanjing, C0009S-1). In brief, B16F10 cells were seeded in 96-well plates, 2–2.5 × 10³ cells per well, and placed in a 37 °C, 5% CO₂ incubator for 24 h. After cell adherence, the FBS was decreased to 2.5%, and treated with different concentrations of apigenin (0~10 µM) and cultured for 48 h or treated with H₂O₂ (0~1 mM) and cultured for 12 h. MTT (0.5 mg/mL, 200 μL/well) treatment was applied, and samples were incubated at 37 °C for 4 h. Then, we removed the solution, dimethyl sulfoxide (DMSO) (150 μL/well) was added, and samples were oscillated gently for 5 min. The absorbance was detected at 570 nm [10].

In order to observe the cells’ morphological change, B16F10 cells were seeded in 10 cm cell culture dishes and treated with various concentrations of apigenin (0.1~5 µM) for 48 h, then treated with H₂O₂ (0.4 mM) for 12 h, and fixed with 3.7% paraformaldehyde for 10 min. Subsequently, refer to the staining instruction of FITC–Phalloidin was purchased from Shanghai Yeasen Biotechnology Co. Ltd. (Shanghai, China). Briefly, the fixed cell samples were stained with the FITC–Phalloidin fluorescence probe for 20~60 min, then washed with PBS 2~4 times, absorbing excess water. Then, the nuclei were stained with DAPI (blue fluorescence) and observed under the fluorescence microscope (Tokyo, Japan, Olympus) [10].

The effect of apigenin on ROS in B16F10 cells under oxidative stress or in zebrafish was detected by fluorescence probe DCFH-DA. Briefly, the cells were seeded on 6-well cell culture plates and treated with various concentrations of apigenin (0.1~5 µM) for 48 h, then treated with 0.4 mM H₂O₂ for 12 h. Zebrafish embryos were treated with egg water containing apigenin (10 µM) for 24 h, then cultured in 0.5 mM H₂O₂ for 4 h. Then, they were stained with the DCFH-DA fluorescence probe to visualize the intracellular ROS and observed by fluorescence microscope (Japan, Olympus). The fluorescence intensity was analyzed by ImageJ software version 1.53t [42].

The Total Superoxide Dismutase (T-SOD, E-BC-K020-M) assay kit, Catalase (CAT) activity assay kit (E-BC-K031-S), and Malondialdehyde (MDA, E-EL-0060) assay kit were purchased from Elabscience (Wuhan, China). The SOD content and CAT and MDA activity were measured according to the instructions of each kit.

The alterations of MMP in B16F10 cells were analyzed by the JC-1 staining assay kit purchased from Biyotime Biotechnology (Nanjing, China, CA1310) according to the instructions. Briefly, the cells were seeded on 6-well culture plates and treated with various concentrations of apigenin (0.1~5 µM) for 48 h, then treated with H₂O₂ (0.4 mM) for 12 h; the cells were washed with PBS and then stained with JC-1 at 37 °C in the dark for 30 min. After being washed with staining buffer twice, they were observed by fluorescence microscope (Japan, Olympus) [43].

Senescence-associated β-galactosidase activity in senescent cells was elevated by a senescence cells histochemical staining kit purchased from Biyotime Biotechnology (Nanjing, China, C0602). Briefly, cells were seeded on 6-well culture plates and treated with various concentrations of apigenin (0.1~5 µM) for 48 h, then cultured with 0.4 mM H₂O₂ for 12 h. Finally, cells were stained according to the manufacturer’s instructions. Senescence-associated β-galactosidase was stained blue. We used a light microscope (400×) to collect images [29].

Total cell protein was obtained according to the above-mentioned method after the determination of the protein concentration by the BCA assay kit purchased from Biyotime Biotechnology (Nanjing, China, P009). Then, the lysates were denatured by heating and boiling at 100 °C for 10 min for Western blot assay. The proteins were separated on 8%, 10%, 12%, and 15% SDS-PAGE gels and then transferred into the polyvinylidene fluoride membranes (PVDF membranes). Subsequently, the PVDF membranes were blocked with 5% skimmed milk at 24 °C for 2 h, washed three times with TBST, then incubated with primary antibodies overnight at 4 °C, and then incubated with secondary antibodies for 2 h at 24 °C. Finally, the PVDF membranes were visualized using an ECL reagent, and detected with a luminous imaging system (Tanon-4160). The results shown here are representative of at least three repeated experiments [10].

All the experiments were repeated three times, and all data are presented as the mean ± standard error of the mean (Mean ± SEM). All were processed by GraphPad prism 9, and statistical analysis was performed with one-way ANOVA (and nonparametric) followed by Turkey’s post hoc test for multiple comparisons. Values of p < 0.05 indicated statistical significance.