*3.3. Effects of FTGML on Intracellular Melanogenesis*

The first step in studies of melanin production is usually to measure intracellular tyrosinase activity, since tyrosinase is the rate-limiting enzyme involved in melanin synthesis [12]. To test the inhibitory effect of FTGML on intracellular tyrosinase activity, B16F10 cells were exposed to FTGML solution in the concentration range of 0~1.6 mg/mL for 48 h. As shown in Figure 3A, FTGML significantly inhibited tyrosinase activity compared

with untreated cells (*p* < 0.05). With the increase of FTGML concentration, intracellular tyrosinase activity decreased gradually. When the FTGML concentration was 1.6 mg/mL, the residual tyrosinase activity was 43.87 ± 5.89% of that of the control. This was not a statistically significant difference (*p* > 0.05) from kojic acid (positive control substance) at 0.75 mg/mL.

**Figure 3.** Effects of FTGML and kojic acid (KA) on melanin content and tyrosinase activity in B16F10 melanoma cells. Cells were treated with various concentrations for 48 h (0–1.6 mg/mL). (**A**) Tyrosinase activity. (**B**) Relative melanin content. Means with different lowercase letters are significantly different (*p* < 0.05) among the different groups.

To further explore the effect of FTGML on melanin production, we detected the residual melanin content in B16F10 cells after FTGML treatment. The results in Figure 3B show that FTGML significantly reduced melanin in B16F10 cells, especially when the FTGML concentration was higher than 0.8 mg/mL. When the concentration of FTGML was 1.6 mg/mL, the intracellular melanin content was 56.44 ± 15.05% of the control group. The content of residual melanin in cells after kojic acid (0.75 mg/mL) treatment was 46.67 ± 6.31% of that of the control group, which was not significantly different from that after FTGML (1.6 mg/mL) treatment (*p* > 0.05). These results suggest that FTGML can achieve whitening by inhibiting the synthesis of melanin in cells, and can achieve a similar effect to kojic acid to a certain extent. This is similar to the results of Han et al., who found that oyster hydrolysate significantly reduced intracellular melanin content [24].

#### *3.4. Effects of FTGML on Intracellular Antioxidant Activity*

In the production of melanin, tyrosine is required to produce dopamine in an oxidized environment and subsequently, to produce dopamine. Therefore, the existence of reactive oxygen species, such as superoxide anion and hydroxyl radical, is conducive to the synthesis of melanin [25]. Studies have shown that peptides with oxygen free radical scavenging ability can inhibit the biosynthesis of melanin in cells [8,26].

Glutathione (GSH) is an important intracellular regulatory metabolite, and its redox states (i.e., reduced glutathione (GSH) and oxidized glutathione (GSSG)) are important for many physiological processes. The effect of FTGML on GSH content in B16F10 cells is shown in Figure 4A. The content of GSH in cells is positively correlated with the concentration of added FTGML. When the concentration of FTGML is 1.6 mg/mL, the content of GSH in cells is 1.78 times higher than that in the blank group. GSH, as a small peptide substance with strong reducibility in cells, can scavenge intracellular free radicals, leading to its critical role in the production of melanin [27]. The effect of FTGML on GSSG content in B16F10 cells is shown in Figure 4B. The addition of FTGML reduced the content of GSSG in B16F10 cells. The GSG:GSSG ratios of B16F10 cells treated with different concentrations of FTGML (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mg/mL) and 0.75 mg/mL kojic acid were 2.10 ± 0.23, 2.57 ± 0.09, 3.61 ± 0.10, 4.79 ± 0.25, 5.00 ± 0.45, 5.22 ± 0.29, 5.68 ± 0.22, respectively. These

**Figure 4.** The effect of FTGML and kojic acid on intracellular antioxidant activity. The GSH contents (**A**), GSSG contents (**B**), ROS levels (**C**), MDA contents (**D**), SOD (**E**), CAT (**F**), and GPX (**G**) activities of B16F10 cells at different treatments. Means with different lowercase letters are significantly different (*p* < 0.05) among the different groups.

It has been shown that ROS play an important role in the regulation of melanogenesis and melanocyte proliferation [28]. ROS scavengers or formation inhibitors can reduce melanogenesis in melanocytes. Malondialdehyde (MDA) is one of the products of cellular membrane lipid peroxidation, and can be used to reflect the extent of oxidative stress damage to cells [29]. To elucidate the protective mechanism of FTGML against oxidative stress in B16F10 cells, the intracellular ROS level and MDA content of B16F10 cells were measured, as shown in Figure 4C,D, respectively. Intracellular ROS and MDA dropped to 62.81% and 50.46% of the untreated group, respectively, as the amount of FTGML was increased. After kojic acid treatment, intracellular ROS and MDA dropped to 58.94% and 51.49% of the untreated group, respectively. This means that after treatment with FTGML (1.6 mg/mL) and kojic acid (0.75 mg/mL), there was no significant difference (*p* > 0.05). This demonstrated that FTGML significantly inhibited intracellular ROS and MDA production, and protected B16F10 cells from oxidative damage. This is similar to the study by Huang et al., who found that [8]-gingerol inhibited melanogenesis in melanoma cells, and that the addition of 100 μM reduced intracellular ROS levels to 71.01 ± 1.45% of the blank group, indicating that it significantly depleted ROS levels in B16F10 cells [30].

SOD, CAT, and GPx are antioxidant enzymes that work together to decrease ROS, and protect cells from oxidative stress damage [31]. It can be seen from Figure 4E,G that the activities of SOD, CAT, and GPx increased by 63.10%, 64.53%, and 69.29%, respectively, after FTGML treatment of B16F10 cells, and increased by 68.51%, 56.00%, and 84.33%, respectively, after treatment with kojic acid. Compared with kojic acid, similar effects to FTGML were observed in SOD and CAT activities (*p* > 0.05). However, there is still a certain gap between the two in GPx activity (*p* < 0.05), and with the increase of FTGML concentration, there is no significant change in GPx activity (*p* > 0.05). The activity of antioxidant enzymes (SOD, CAT, and GPx) significantly affects the sensitivity of the skin to oxidative damage, including skin pigmentation problems [32]. Therefore, FTGML shows a protective effect on oxidation by increasing the activity of antioxidant enzymes in B16F10 cells, thereby avoiding melanin deposition. Some researchers' reports also indicate that peptides interfere with skin biochemical reactions by protecting cells against oxidative damage, for example, sorghum kafrins-derived peptide fraction [26], and this is mainly due to their ability to enhance the activity of antioxidant enzymes in the cell.

#### *3.5. Effects of FTGML on the Melanogenesis-Related Signaling Pathway in B16F10 Melanoma Cells*

In this study, we examined the expression of melanogenesis-related proteins, including MITF and cAMP, and melanogenesis-regulating molecules, including PI3K/AKT, p38, and JNK, to elucidate the potential mechanisms by which FTGML inhibits melanogenesis in B16F10 melanoma cells. The results are shown in Figure 5.

cAMP is a well-known intracellular second messenger, and its mediated signaling is the main cascade of melanin production, which is largely influenced by changes in intracellular cAMP levels [12]. As shown in Figure 5A, FTGML (1.6 mg/mL) and kojic acid (0.75 mg/mL) reduced intracellular cAMP levels in B16F10 cells by 48.77% and 45.38%, respectively, compared to untreated controls (*p* < 0.05). cAMP is an important mediator of intracellular signal-activated protein kinase A (PKA). In the absence of cAMP, PKA is inactive, and its two PKA catalytic subunits bind to the two PKA regulatory subunits. When cAMP is present, cAMP binds to the regulatory subunits of PKA, and induces dissociation of the PKA catalytic subunits from the PKA holoenzyme complex, and the released PKA catalytic subunits are activated, ultimately activating the cAMP response element binding protein (CREB), and thus promoting melanin synthesis [33]. Therefore, it can be concluded that FTGML treatment reduces the intracellular cAMP level, which is beneficial to the regulation of subsequent signaling pathways. Similar results were found by Han et al. Oyster hydrolysate exhibited anti-melanogenic activity by downregulating the cAMP signaling pathway, thereby reducing melanin synthesis [24]. Phosvitin-derived peptide Pt5 was shown to be involved in the cAMP pathway to inhibit melanogenesis, with no significant effect on the Wnt and MAPK signaling pathways [34].

**Figure 5.** The effect of different concentrations of FTGML on the expression level of related proteins. 0.1, 0.2, 0.4, 0.8, and 1.6 represent the concentration of FTGML (mg/mL), KA represents kojic acid, and the concentration is 0.75 mg/mL. The cAMP contents (**A**), western blot analysis. (**B**), the relative protein expression was examined by western blot analysis (**C**), the relative protein expression of MITF was examined by western blot analysis (**D**). Means with different lowercase letters are significantly different (*p* < 0.05) among the different groups.

cAMP can also regulate melanogenesis through PKA non-dependent mechanisms, such as phosphatidylinositol-3-kinase (PI3K). One of the key effectors of PI3K is protein kinase B (Akt). The results shown in Figure 5C were obtained by analysis of Figure 5B. Both FTGML and kojic acid treatment decreased the expression of p-PI3K and p-Akt compared to untreated controls. p-PI3K/PI3K and p-Akt/Akt ratios of B16F10 cells were 0.67 and 0.89, respectively, after 1.6 mg/mL FTGML treatment. In addition, 0.75 mg/mL kojic acid treatment decreased the p-PI3K/PI3K and p-Akt/Akt ratios to 0.59 and 0.77, respectively. Under the stimulation of external signals and intracellular cAMP, activated Akt enhances the binding of MITF to the M-box by phosphorylating glycogen synthase kinase 3β (GSK3β), and promotes its loss of activity. The reduction in GSK3β activity enhances the binding of MITF to the M-box, and synergistically stimulates the tyrosinase promoter with MITF, enhancing its binding to the tyrosinase promoter, and thus, promoting melanogenesis [35]. The results showed that FTGML reduced the phosphorylation level of PI3K/Akt by downregulating cAMP. It has been shown that diosgenin has the effect of regulating the reduction of PI3K expression and phosphorylation of AKt, which affects the phosphorylation of downstream GSK-β, which, in turn, allows the downregulation of MITF expression, leading to a reduction in melanin synthesis [36].

In addition to the cAMP-mediated signaling pathway described above, there is also the mitogen-activated protein kinase (MAPK) signaling pathway, which is involved in the expression and activation of MITF [37]. Based on the apoptotic results in Section 3.2, we speculate that it may be FTGML that promotes apoptosis in B16F10 cells, resulting in reduced melanin. Several past studies have shown that JNK and p38 are the two most important factors in MAPK that affect apoptosis [9,38,39]. As shown in Figure 5C, both FTGML and kojic acid treatment reduced the expression of p-JNK and p-p38 compared to untreated controls. p-JNK/JNK and p-p38/p38 ratios of B16F10 cells after 1.6 mg/mL FTGML treatment were 0.65 and 0.70, respectively. In addition, 0.75 mg/mL kojic acid treatment reduced the p-JNK/JNK and p-p38/p38 ratios of B16F10 cells, which had p-JNK/JNK and p-p38/p38 ratios of 0.68 and 0.63, respectively. It has been reported that the inhibition of the JNK pathway reduces melanin production [40], and that phosphorylation of p38 activates MITF expression and upregulates melanogenesis-related proteins, which, in turn, affects melanin synthesis [41]. These results suggest that FTGML inhibits melanin synthesis through downregulation of the JNK and p38 signaling pathways.

MITF is located downstream of these signaling pathways and upstream of tyrosinase, and is involved in the activation of several melanocyte-related genes. It regulates both tyrosinase and its associated proteins on melanin synthesis, as well as the growth, development, and differentiation of melanocytes [42]. As shown in Figure 5D, the expression of MITF was significantly reduced after FTGML acted on melanocytes, and the inhibition was most evident at the highest concentration of 1.6 mg/mL of FTGML, which was consistent with our prediction, indicating that FTGML could reduce the expression of MITF. It was also verified that inhibition of the above signaling pathways (cAMP-PI3K/Akt and MAPK pathways) also significantly affected the expression of MITF. Based on these results, we hypothesize that FTGML downregulates the phosphorylation of PI3K/Akt in this signaling pathway by decreasing cAMP levels. It also downregulates the phosphorylation of JNK and p38 in the MAPK signaling pathway. Together, these two pathways are involved in FTGML's inhibition of MITF expression-mediated melanogenesis, with the PI3K signaling pathway being relatively more important. Of the available studies on tyrosinase inhibitory peptides, very little has been done on their signaling pathways. Peptides derived from the fermented microalga (*Pavlova lutheri*) [8] and oyster hydrolysate [24] have been demonstrated, but they have all been studied by selecting one of the signaling pathways. The findings of the present study suggest the possibility that multiple signaling pathways may still act together in melanin inhibition.

### *3.6. Principal Component (PCA), Cluster Analyses (HCA), and Correlation Analysis*

Principal component analysis was performed on 10 indicators from 6 samples with different FTGML additions using SPSS 20.0 software. As shown in Table 1, a total of two principal components were extracted, and the cumulative value of the contribution of the principal components reached 94.836%, which can explain the vast majority of the original information. Principal component 1 is the most important, with a contribution of 87.144% of the variance, which can represent 87.144% of the total information. Principal component 2 has a variance contribution of 7.692%.

The corresponding eigenvectors and load matrices of the principal components are shown in Table 2. It can be concluded that PC1 is associated with 15% melanin, 17% cAMP, 15% GSH, 26% CAT, 23% MDA, 15% ROS, and 44% SOD, whereas PC2 is mainly associated with 34% tyrosinase, 44% GPx, and 57% GSSG. Using PC1 as the *x*-axis, and PC2 as the *y*-axis, a plot was drawn based on the corresponding load values, as shown in Figure 6A. Ten indicators were scattered in the first and third quadrants of the axes. The linear equation of the combined score of each principal component was derived from the eigenvector matrix of the two principal components, and the relative contribution of the variance corresponding to each principal component was used as the weight to build the comprehensive evaluation model: F1 = 0.154 × 1 + 0.170 × 2 − 0.154 × 3 − 0.146 × 4 − 0.366 × 5 − 0.259 × 6 + 0.238 × 7 + 0.225 × 8 + 0.147 × 9 − 0.442 × 10; F2 = −0.011 × 1 −

0.024 × 2 + 0.342 × 3 − 0.001 × 4 + 0.567 × 5 + 0.130 × 6 − 0.436 × 7 − 0.089 × 8 + 0.005 × 9 + 0.372 × 10. Based on the 53.110% of principal component 1, and 41.727% of principal component 2, the composite score function can be derived as F = 0.53110 × F1 + 0.41727 × F2. The composite score of each sample was calculated and ranked by the above model (Table 3). The F1 and F2 scores for each sample are represented as coordinates in Figure 6A. Combined with the PCA composite evaluation model scores, the samples were further classified into four groups by hierarchical cluster analysis (HCA) (Figure 6B-1): control (0), low performing samples (0.1 and 0.2), mild samples (0.4), and high performing samples (0.8 and 1.6). The classification results indicated that FTGML had good activity at 0.8 mg/mL and 1.6 mg/mL addition, and was a suitable anti-melanin agent and antioxidant. The 10 indicators were systematically clustered, and the results are shown in Figure 6B-2, which can be divided into two major categories and four minor categories, which is consistent with what is shown in Figure 6A. Among the subcategories, the antioxidant indicators that were in the same category as the anti-melanin indicators were MDA and ROS.

**Table 1.** Characteristic values and contribution rates of principal components.


The correlation between anti-melanin properties and other antioxidant properties is shown in Figure 6C. Melanin content was positively correlated with cAMP and tyrosinase activity (both greater than 0.85), indicating a strong correlation between these three factors. GSH and ROS were negatively/positively correlated with melanin (−0.90 for GSH and 0.96 for ROS), indicating that an increase in GSH and a decrease in ROS favored a reduction in melanin content, which was related to the free radical scavenging capacity of GSH, and the fact that a decrease in ROS inhibited the melanin production process. MDA was positively correlated with melanin (0.95), suggesting that a reduction in the content of lipid peroxidation products in the cell membrane favors a reduction in melanin content. A strong correlation between MDA and ROS (0.99), and a negative correlation between CAT and melanin (−0.95), suggest that reactive oxygen species affecting the cell can directly regulate

melanin production. Overall, GSH, CAT, MDA, and ROS are the main antioxidant factors influencing melanin production.

**Figure 6.** Principal component analysis (**A**) loading plot and score plot. (**B-1**) Hierarchical cluster analysis under different FTGML additions. (**B-2**) Hierarchical cluster analysis of various indicators under different FTGML additions. (**C**) Results of correlation analysis. melanin: mean melanin content (% of control); cAMP: mean cAMP content (nmol/L); tyrosinase: mean tyrosinase ability (% of control); GSH and GSSG: mean GSH and GSSG content (μg/mg protein), respectively; MDA: mean MDA content (nmol/mg protein); ROS: mean ROS content (IU/mL); CAT, GPx, and SOD: mean CAT, GPx, and SOD content (μg/mg protein), respectively.


**Table 2.** Eigenvectors and load matrices corresponding to principal components.

**Table 3.** Principal component score, comprehensive score, and ranking.


#### **4. Conclusions**

This study investigated the effects of tyrosinase inhibitory peptide FTGML, derived from grass carp fish scale gelatin, on the melanin inhibitory signaling pathway and intracellular antioxidant activity in murine B16F10 melanoma cells. The results showed that FTGML significantly inhibited intracellular tyrosinase activity and melanin content, and had a positive effect on intracellular antioxidant activity. There was a strong correlation between MDA, ROS, GSH, and CAT among the antioxidant indicators for their ability to counteract melanin, which provides theoretical support for predicting the anti-melanin ability or antioxidant activity. In addition, two signaling pathways through which FTGML affects melanin synthesis were revealed. FTGML downregulates MITF expression by inhibiting the cAMP-PI3K/Akt signaling pathway, as well as p38 and JNK in the MAPK signaling pathway. On the other hand, p38 and JNK were found to be present for the first time in the peptide mediated melanin synthesis signaling pathway, whereas this signaling pathway and the STAT3 factors also promoted apoptosis in B16F10 cells. These results suggested that FTGML can reduce melanin production in mouse B16F10 melanoma cells.

**Author Contributions:** Conceptualization: Z.H., X.S. and Z.T.; Methodology: Z.H.; investigation: Z.H., L.Z. and S.H.; resources: Z.T.; supervision: X.S. and Z.T.; writing—original draft: Z.H.; writing review and editing: X.S. and Z.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Key R&D Program of China (No. 2018YFD0901101), and the Key R&D Program of Jiangxi Province (No. 20192ACB60005), National Natural Science Foundation of China (No. 32160576, 31760445).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
