Canines skin cell lines PETCC167 and cats skin cell lines PETCC172 were purchased from the PETCC (Nourse, Wuhu, China) and were cultured in DMEM medium containing FBS (10%) and penicillin/streptomycin (P/S, 1%). PETCC 167 is a canine skin tumor cell collected by PETCC in 2023, it is extracted from a 10-year-old female Toy Poodle. PETCC 172 is a feline skin tumor cell collected by PETCC in 2023, it is extracted from an 8-year-old male American Shorthair. All cells were cultured in a 37°C constant temperature incubator containing 5% CO₂.

Cells were inoculated into 96-well plates with a density of 2,000 cells per well. Subsequently, cells were treated with keratin hydrolyzed (2 mg/mL), egg yolk lecithin (0.25 mM), cysteine (1 mM), fish collagen peptide (5 mg/mL), and cow bone collagen protein (5 mg/mL) for 24 h, 48 h or 72 h, and the OD value was measured at each time point. After incubation with CCK8 reagent (Meilunbio, MA0218) at 37°C for 2 h, the absorbance at 450 nm was measured with Multiscan Spectrum (Thermo) every day to detect cell proliferation.

Cell lysate proteins were collected and loaded on SDS polyacrylamide gels as previously described (17). Membranes were incubated with appropriate antibodies against β-catenin (51067-2-AP, Proteintech, 1:2000), β-Actin (66009-1-Ig, Proteintech, 1:20000), Tublin (14555-1-AP, Proteintech, 1:1500) or H3 (17168-1-AP, Proteintech, 1:8000) at 4°C overnight. After washes with PBST saline membranes were incubated with a horseradish peroxidase–linked secondary antibody (1:5000) for 1 h. Immunoreactive bands were detected with an enhanced chemiluminescent plus reagent kit.

RNA purification kit (EZBioscience) and Reverse Transcription Master Mix (EZBioscience) were used to extract total RNA from cells and perform reverse transcription reactions, respectively. Perform qRT-PCR experiment using Applied Biosystem 7,300 plus Sequence Detection System (Applied Biosystems). The endogenous controls for mRNA was β-actin RNA. The relative quantification (2^−ΔΔCT) method was used for results analyzing. Primers were shown in Supplementary Table S1. The qPCR reaction procedure was shown in Supplementary Table S2.

Separate the nucleus and cytoplasm of cells using the nuclear protein and cytoplasmic protein extraction kit (Beyotime, P0027), and prepare whole cell lysate for WB. All operations are carried out according to the instructions.

The experimental data represent the mean ± standard error of three repeated experiments. Statistical software GraphPad prism 9.0 was used for statistical analysis. Comparisons between two groups for difference analysis were performed with two-tailed Student t-test, significance level were indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significance.

Maintaining the proliferation ability of skin cell lines is a fundamental condition for hair regeneration. To investigate the effects of several nutrients on the proliferation of skin cell lines in canines and cats, we selected five nutrients including keratin hydrolyzed, egg yolk lecithin, cysteine, fish collagen peptide, and cow bone collagen protein. The results showed that keratin hydrolyzed (Figure 1A), egg yolk lecithin (Figure 1B), cysteine (Figure 1C), and fish collagen peptide (Figure 1D) significantly promoted the proliferation of skin cell lines in canines and cats, while cow bone collagen protein did not affect cell proliferation (Figure 1E). In summary, keratin hydrolyzed, egg yolk lecithin, cysteine and fish collagen peptide are beneficial for maintaining the proliferation ability of skin cell lines in canines and cats.

Given the crucial role of the Wnt/β-catenin signaling pathway in the proliferation and hair regeneration of skin cell lines, we next examined the effects of several nutrients on the β-catenin protein levels in canines and cats skin cell lines. The results showed that keratin hydrolyzed (Figure 2A), egg yolk lecithin (Figure 2B), and fish collagen peptide (Figure 2D) significantly upregulated the protein levels of β-catenin in the canines and cats skin cell lines. However, cysteine selectively affects the protein levels of β-catenin in canines skin cell line, while exhibiting no impact on skin cell line (Figure 2C). Although our results in Figure 1 have shown that cysteine promotes the proliferation of skin cell lines in canines and cats, this effect is not achieved by promoting β-catenin expression. In summary, keratin hydrolyzed, egg yolk lecithin and fish collagen peptide upregulated the protein levels of β-catenin.

When the Wnt/β-catenin signal is activated, the β-catenin protein enters the nucleus to promote the expression of target genes, which helps with hair regeneration in hair follicles (11, 18). Our results have demonstrated that keratin hydrolyzed, egg yolk lecithin, and fish collagen peptide upregulate the protein level of β-catenin, but it is unclear whether these nutrients can promote the protein level of β-catenin in the nucleus. Next, we examined the localization of β-catenin in the nucleus of skin cell lines from canines and cats treated with keratin hydrolyzed, egg yolk lecithin, and fish collagen peptide. Our results demonstrated that keratin hydrolysate treatment enhanced β-catenin expression in both nuclear and cytoplasmic compartments of PETCC167 and PETCC172 cells (Figures 3A,B). In PETCC167 cells, egg yolk lecithin specifically increased nuclear β-catenin expression without affecting its cytoplasmic levels (Figure 3C). However, in PETCC172 cells, egg yolk lecithin treatment led to elevated β-catenin expression in both nuclear and cytoplasmic fractions (Figure 3D). Similarly, fish collagen peptide selectively enhanced nuclear β-catenin expression in PETCC167 cells (Figure 3E), while in PETCC172 cells, it increased β-catenin levels in both cellular compartments (Figure 3F). Collectively, these findings suggest that keratin hydrolysate, egg yolk lecithin, and fish collagen peptide promote β-catenin nuclear translocation.

When β-catenin enters the nucleus, it causes the expression of target genes (19, 20). Our results have shown that keratin hydrolyzed, egg yolk lecithin, and fish collagen peptide significantly promote the nuclear translocation of β-catenin, but it is unclear whether this leads to an increase in its target gene expression. Subsequently, we examined the expression levels of target genes of β-catenin, including c-Myc, CCND1, Axin2, Tcf7, Sox21, and Lef1 in canines and cats skin cell lines treated with keratin hydrolyzed, egg yolk lecithin, and fish collagen peptide. Compared to the control group, keratin hydrolysate treatment significantly upregulated β-catenin target genes in both PETCC167 and PETCC172 cells, with Axin2 and Tcf7 showing the most pronounced elevation in PETCC167 cells, while Sox21 and Lef1 exhibited the highest upregulation in PETCC172 cells (Figures 4A,B). Similarly, egg yolk lecithin treatment demonstrated comparable trends in both cell lines, with Lef7 displaying the most significant upregulation in PETCC172 cells (Figures 4C,D). Consistent with these observations, fish collagen peptide treatment also enhanced the expression of these target genes, particularly Lef7 in PETCC167 cells, while Sox21 and Lef1 showed the most substantial increases in PETCC172 cells (Figures 4E,F). Furthermore, treatment with Wnt/β-catenin inhibitor KYA1797K partially reversed the upregulation of target genes induced by nutrients to varying extents, indicating that nutrients enhance gene expression levels through the Wnt/β-catenin signaling pathway (Figures 4A–F). It is worth noting that the upregulation of c-Myc and CCND1 expression is consistent with the promotion of cell proliferation. More importantly, the Axin2, Tcf7, Sox21, and Lef1 are crucial for hair follicle regeneration, indicating that keratin hydrolyzed, egg yolk lecithin, and fish collagen peptide may play a role in hair follicle regeneration in canines and cats.