Roles of bovine sialoglycoproteins for anti‐skin aging and accelerating skin wound healing

Fibroblasts are the most predominant cell subpopulation in the dermal layer of human skin, they play an important role in maintaining skin architecture and function. The senescence of fibroblasts is one of major causes of skin aging and chronic wound in the elderly, which is accompanied with a reduction of α2,6‐sialylation on the cell surface.


| INTRODUC TI ON
Skin is the largest organ covering the entire surface of the body.It provides a protective physical barrier between the body and the environment, preventing loss of water and electrolytes, reducing penetration by chemicals, and protecting against pathogenic microorganisms. 1 The dermis is a major contributor to skin thickness, elasticity, and water content. 2 Fibroblasts are the main resident cell type of the dermal layer, which play a vital role in skin architecture and function.Decreased activity and cellular dysfunction of dermal fibroblasts are the main causes of skin aging or disorders.For example, the ability of dermal fibroblasts to proliferate and secrete extracellular matrix proteins was decreased in aged skin, causing structural changes in the dermis and ultimately leading to the appearance of skin aging features, such as wrinkles and reduced elasticity. 3,4Moreover, the cellular dysfunction of dermal fibroblasts, such as impaired migration, is a major cause of chronic wound in diabetic patients. 5][8][9][10][11] As the largest organ in the body, the skin shows more obvious, and visible signs of aging than other internal organs when one becomes older.Although skin aging is inevitable, it can be somewhat delayed.Therefore, many people need to spend a considerable amount of daily effort on cosmetics or pharmaceuticals attempting to prevent or reverse skin aging, this huge demand continuously stimulates research on skin aging and its treatment. 12,13The senescence of dermal fibroblasts is one of the direct causes of skin aging due to their important role in the synthesis and degradation of the extracellular matrix. 14Moreover, autologous fibroblast cell therapy has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of facial skin wrinkles, and it is more effective than traditional dermal fillers for treating nasolabial wrinkles. 15Consequently, enhancing the viability of the dermal fibroblasts may be a more effective way to delay skin aging than conventional methods.
In addition to the important role in cosmetology, establishing a protective barrier between the inside and outside of the body is another essential function of the skin.Once the skin is injured, it needs to be repaired immediately to restore the skin's integrity in order to re-establish homeostatic mechanisms.Typically, cutaneous wound healing is divided into four successive but overlapping phases namely hemostasis, inflammation, cellular proliferation, and remodeling. 16Dermal fibroblasts play a prominent role during these processes.They proliferate and invade the lesion site, regulate local inflammation, and generate ECM as a scaffold for other cells, they may also differentiate into myofibroblasts to promote wound contraction. 17Deregulation of fibroblast proliferation, differentiation, apoptosis as well as ECM secretion during wound healing will lead to ulcerative skin defects (chronic wounds) or scar formation (hypertrophic scars and keloid). 5It has been proven that the dermal fibroblasts in ulcer wounds exhibit senescence and diminished migratory capacity. 18e surface of human cells is covered with a variety of glycans linked to proteins or lipids, which are involved in many biological processes, such as intercellular communication, viral infection, tumorigenesis, and aging. 19Dynamic changes of glycans on the cell surface are relevant to cell growth, development, and aging processes.Sialic acid as a monosaccharide frequently appears at terminal of glycans, and its alterations are more susceptible to cell status.The reduction of sialylation in human senescent cells was first reported in 1979. 20rrently, it has been demonstrated that both α2,6-and α2,3sialylation on the surface of dermal fibroblasts was reduced during the human aging. 21Reduced sialylation on the cell surface could inhibit the differentiation of fibroblasts to myofibroblasts, leading to the delayed wound healing in the elderly. 22Therefore, modulating sialylation on the fibroblast surface is an attractive strategy in cosmetic and skin care.
Previously, we have purified sialoglycoproteins from bovine milk using serotonin-functionalized magnetic particles and validated their ability against influenza A virus. 23In the present study, we investigated the effects of bovine sialoglycoproteins on cultured normal human dermal fibroblasts (NHDF).The proliferation and migration of NHDF cells were examined using CCK-8 and wound scratch assays, respectively.Wound healing was assessed using an in vitro wound healing model (fibroblast-populated collagen lattice, FPCL).Then, the expression of basic fibroblast growth factor (FGF-2) and transforming growth factor β1 (TGF-β1), which play key roles in wound healing and scar formation, respectively, was examined by RT-qPCR and ELISA assays.Subsequently, the production of type I collagen, which is a direct contributor of scar formation, was also examined.Furthermore, the effects of bovine sialoglycoproteins on sialylation of NHDF cells was examined using lectin histochemistry and lectin blotting assays, and the results was further confirmed by the changes of the relevant sialyltransferase expression.In summary, this study provides new ideas for skin anti-aging and accelerating skin wound healing, and bovine sialoglycoproteins may be developed as a skin anti-aging agent or as a candidate for accelerating skin wound healing.

| Bovine sialoglycoprotein purification and cell culture
Bovine sialoglycoproteins were purified from bovine milk according our previously report. 23NHDF cells were purchased from BeNa culture collection, which were isolated from adult human skin and cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone) containing 10% (vol/vol) fetal bovine serum (FBS, GIBCO), 100 U mL −1 penicillin, and 100 μg/mL streptomycin at 37°C under 5% CO 2 .For bovine sialoglycoproteins treatment, NHDF cells were first cultured in DMEM medium with 10% FBS.After 24 h of culture, the medium was replaced with serum-free DMEM containing bovine sialoglycoproteins and the culture was continued.Control group was cultured in serum-free DMEM.

| Determination of cell doubling time
NHDF cells were routinely seeded in 6-well plate at the density about 5 × 10 4 cells/well.After 72 h of bovine sialoglycoproteins (0.5 mg/mL) treatment, the cultured cells were harvested and counted.Cell doubling time was calculated using the following formula: Dt = t/(log 2 X − log 2 Y), where Dt = doubling time, t = time of culture, X = number of cells after t time of culture, Y = number of cells seeded at the beginning.

| Scratch wound healing assay
Scratch wound healing experiment was performed according to the protocol reported in previous literature. 24In brief, NHDF cells were seeded in 6-well plate at a density about 5 × 10 4 cells/well and cultured in DMEM with 10% FBS.When the cells reached confluence, a straight scratch was made across the cell monolayer with a white pipette tip.After removal of culture medium and detached cells, remaining cells were cultured in serum-free DMEM with 0.5 mg/mL of bovine sialoglycoproteins.The cell images were acquired at 0, 12, 24, 36, 48, 60, and 72 h of treatment under a microscope.The area of the scratches was measured using ImageJ software.The wound closure was calculated using the following equation: wound closure % = ((A t=0h −A t=Δh )/A t=0h ) × 100%, where A t=0h is the area of the wound measured immediately after scratching (time zero), and A t=Δh is the area of the wound measured after h hours of the scratching.

| Contraction of fibroblast-populated collagen lattice
FPCLs was prepared using rat tail tendon collagen (Solarbio) according to the manufacturer's instructions.FPCL was directly incubated in serum-free DMEM with 0.5 mg/mL bovine sialoglycoproteins.After 0, 12, 24, 36, 48, and 60 h of incubation, the images of FPCLs were acquired and the area of FPCLs was measured using ImageJ software.The contraction rate of the FPCLs was calculated using the following formula: contraction rate (%) = ((A t=0h −A t=Δh )/A t=0h ) × 1 00%, where A t=0h is the area of FPCLs at the beginning (time zero), A t=Δh is the area of FPCLs measured after h hours of incubation.

| Reverse transcription-quantitative real time PCR (RT-qPCR)
NHDF cells were first treated with bovine sialoglycoproteins (0.5 mg/mL) for 48 h.Then, total RNA of cells was extracted using TRIzol reagent (Invitrogen) and subjected to reverse transcription using Hifair® III first Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen) according to the manufacturer's instructions.The RT-qPCR amplification mixtures (total volume, 20 μL) contained 10 μL of Hieff® qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen), 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 1 μL of cDNA template, and 8.2 μL of RNase-free water.qPCR was performed using ViiA™ 7 Real-Time PCR System (Applied Biosystems) using the following amplification conditions: pre-incubation at 95°C for 5 min, 40 cycles of 95°C for 10 s, annealing at 60°C for 20 s, and elongation at 72°C for 20 s.The primers used in this study were synthesized by Tsingke Biotechnology Co., Ltd., and their sequences are shown in Table 1.GAPDH was used as a reference gene.Target gene expression was normalized against expression of GAPDH.Relative quantization was preformed using the comparative ΔΔC t method.

| Enzyme-linked immunosorbent assay (ELISA)
NHDF cells were treated with 0.5 mg/mL bovine sialoglycoproteins for 48 h.The contents of COL-I, TGF-β1, and FGF-2 in culture supernatants were quantified using the Human Type I Collagen ELISA Kit (Lianshuo Biological Technology Co., LTD), Human TGF-β1 ELISA Kit (Beyotime Biotechnology), and Human FGF-2 ELISA Kit (MEIMIAN), respectively, according to the manufacturer's instructions.

| Lectin histochemistry assay
Lectin histochemistry was used to determine the expression of sialic acid on the cells according to our previously reported method. 23iefly, NHDF cells were inoculated into confocal culture dishes (JingAn) and treated with 0.5 mg/mL of bovine sialoglycoproteins for 48 of culture.Then, the cells were sequentially washed with PBS, immobilized with 4% paraformaldehyde for 15 min and blocked with 1× Carbo-Free blocking solution (Vector Laboratories) for 1 h at TA B L E 1 The primer sequences used in the RT-qPCR.

| SDS-PAGE and lectin blotting
NHDF cells were first treated with 0.5 mg/mL of bovine sialoglycoproteins for 48 h.Then, the membrane proteins were extracted using Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific).Protein concentrations were determined using BCA Protein Assay Kit (Beyotime Biotechnology).SDS-PAGE and lectin blot analysis were performed according to the method described previously. 23In brief, 10 μg protein samples were subjected to 10% SDS-PAGE and then stained with silver nitrate.For lectin blot analysis, 20 μg protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore), followed by blocking with 1 × Carbo-Free blocking solution for 1 h at room temperature, and then incubated with Cy5 labeled MAL-II or SNA (10 μg/mL in Carbo-Free Blocking Solution) overnight at 4°C with gentle shaking protected from light.After washing with PBST (0.05% Tween-20 in PBS), the membranes were scanned using a Storm 480 molecular imager (Molecular Dynamics) at a wavelength of 635 nm.

| Western blot assay
NHDF cells were first treated with 0.5 mg/mL of bovine sialoglycoproteins for 48 h.After that, the cells were washed twice with cold and incubated with an anti-ST6GAL1 antibody (Abcam, 1:1000) or anti-beta tubulin antibody (Abways, 1:3000) at 4°C overnight.After washing with PBST, the membrane was exposed to the appropriate horseradish peroxidase-conjugated secondary antibody (Abways, 1:5000) for 1 h at room temperature.After washing again with PBST, the reactive protein bands were visualized by chemiluminescence using an ECL kit (Millipore) and detected using ChemiScope 6200 System (Clinx).

| Statistical analysis
All data were acquired from at least three independent experiments and reported as the mean ± standard deviation (SD).The statistical significances of differences were assessed by analysis of one-way ANOVA followed by Tukey's multiple comparison test or the student's t-test using the GraphPad Prism ver 8.0 software.A value of p < 0.05 was considered statistically significant.

| Bovine sialoglycoproteins promoted the proliferation of NHDF cells
To evaluate the effects of bovine sialoglycoproteins on proliferation of NHDF cells, NHDF cells were cultured in DMEM medium with or without bovine sialoglycoproteins.The viability of cultured cells was measured by CCK-8 assays.The results showed that the cell viability of NHDF cells was enhanced with increasing concentrations of bovine sialoglycoproteins in the range of 0.05 mg/mL-0.5 mg/ mL (Figure 1A).Moreover, bovine sialoglycoproteins at 0.5 mg/mL showed stronger increase in the cell viability than raw milk proteins at the same concentration (Figure 1B).As for the morphology of cultured NHDF cells, bovine sialoglycoproteins treated NHDF cells showed a spindle-like appearance while the untreated cells showed a spherical shape (Figure 1C).In particular, 0.5 mg/mL of bovine sialoglycoproteins significantly shortened the doubling time of NHDF cells compared with the control (37.9 ± 2.7 h vs. 31.1 ± 1.0 h, p < 0.05) (Figure 1D).These results demonstrated that bovine sialoglycoproteins were able to promote the proliferation of NHDF cells.

| Bovine sialoglycoproteins promoted the migration of NHDF cells
The migration ability of bovine sialoglycoproteins-treated and untreated cells was assessed using a scratch wound model.The results (Figure 2) showed that 0. (p < 0.001), respectively.These results suggested that the bovine sialoglycoproteins treatment could improve the migration capability of NHDF cells.

| Bovine sialoglycoproteins accelerated the contraction of FPCL
To evaluate the effect of bovine sialoglycoproteins on skin wound healing, FPCL was prepared and used as an in vitro skin wound model.The results showed that the contraction of FPCL incubated in serum-free DMEM containing 0.5 mg/mL of bovine sialoglycoproteins was more rapidly compared with the control (Figure 3A).In particular, there was a significantly difference in FPCL contraction rate (%) at 24, 36, and 48 h of incubation between the treated and control groups, which was 68.6 versus 59.5 (p < 0.01), 74.5 versus 59.5 (p < 0.001) and 78.6 versus 70.4 (p < 0.05), respectively (Figure 3C).
In addition, NHDF cells in FPCLs treated with bovine sialoglycoproteins showed a better growth state and formed a denser reticular architecture compared to the control (Figure 3B).these results in-

| Effects of bovine sialoglycoproteins on secretion of COL-I, TGF-β1, and FGF-2
COL-I is the most abundant extracellular matrix protein in the dermal layer of skin, and its expression is mainly promoted by TGF-β1. 2 As shown in Figure 4, the expression of COL-I and TGF-β1 in NHDF cells treated with 0.5 mg/mL of bovine sialoglycoproteins was consistently decreased at both the gene and protein levels, and their contents were 0.12 ng/mL and 43.29 pg/mL in the cultured supernatant of the treated group, compared with 0.37 ng/ mL and 66.15 pg/mL of the control, respectively.However, bovine sialoglycoproteins treatment could remarkedly increase the expression of FGF-2 in NHDF cells, and its contents in the cultured supernatants of the treated and control groups were 594.33 pg/ mL and 516.71 pg/mL, respectively.These results demonstrated that bovine sialoglycoproteins treatment could suppress the

| DISCUSS ION
Aging is unavoidable but can be somewhat delayed.Fibroblasts are the most predominant cell subpopulation in the dermis, their capacity to proliferate and secrete ECM plays an important role in skin aging.It has been showed that the proliferation capacity of fibroblasts from elderly were reduced. 3 In addition to maintaining the skin architecture, dermal fibroblasts also play an important role in skin wound healing.Improving the capacity of fibroblasts to migrate and proliferate is beneficial for skin wound healing, however this often result in abnormal scar formation characterized by excessive deposition of collagen within the wound site. 25,26Hence, the ideal treatment for skin wound is to improve the ability of fibroblasts to proliferate and migrate, but decrease the secretion of collagen.TGFβ superfamily is an important mediator of wound healing, which exerts pleiotropic effects by regulating cell proliferation and migration, differentiation, ECM production, and immune modulation. 7In particular, TGF-β1 may regulate COL-I biosynthesis and promote scarring. 8,14,27Absence of Smad3, a direct downstream transcriptional factor of TGF-β1, was sufficient to enhance wound healing and attenuate glial scar formation after stab wound injury to the cerebral cortex of mice.there is now considerable evidence showing that FGF-2 accelerates healing of a wide range of wounds, including pressure ulcers, second degree burns, leg ulcers, and diabetic ulcers, in addition to prevention of scar formation. 10,32These findings support that bovine sialoglycoproteins could be developed as candidates for accelerating skin wound healing and inhibiting scar formation because of their ability to promote FGF-2 expression and inhibit COL-I production.
Glycosylation is one of the most common post-translational modifications of proteins.33,34 As sialic acid is located at the terminal position of glycans, changes in sialylation of N-glycans are more pronounce during aging.For skin fibroblasts, it has been shown that the late passaged cells and the cells obtained from the elderly display reduced α2,6-and α2,3-sialylation, and preferentially reduced cell surface α2,6-sialylation during cellular senescence. 21,35reover, studies have also demonstrated that reduced α2,6sialylation impaired the proliferative capacity of the cells. 24,36These results suggested that the sialylation, especially α2,6-sialylation on the cell surface, is positively correlated with the proliferative capacity of fibroblasts.Therefore, increasing α2,6-sialylation on the fibroblast surface may delay skin aging.8][39] On the other hand, galactose exposure could also activate dendritic cells and macrophages by binding to C-type lectins on the cell surface, leading to a low-grade inflammation, and eventually to chronic disease in the elderly. 40,41This study demonstrated that bovine sialoglycoproteins could increase α2,6-sialylation on the surface of NHDF cells, suggesting that it may have the capability to reduce chronic inflammation in the elderly.
In summary, the present study showed that bovine sialoglycoproteins promoted the proliferation and migration of NHDF cells, accelerated FPCL contraction, increased FGF-2 expression but suppressed TGF-β1 and COL-I production in NHDF cells, and also enhanced α2,6-sialylation on the cell surface.These results suggested that bovine sialoglycoproteins may be a candidate for antiskin aging and scarless wound healing.
photographed under the merged channels of Cy5 and DAPI using a laser scanning confocal microscope (Leica).
PBS and then lysed in ice-cold RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Sigma-Aldrich).Protein concentration was determined by BCA assay.20 μg protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes.The membranes were blocked with 5% Difco™ skim milk (BD Biosciences)

F I G U R E 1
Effect of bovine sialoglycoproteins on the proliferation of NHDF cells.(A) Effect of bovine sialoglycoproteins treatment on the viability of NHDF cells.(B) Effect of 0.5 mg/mL of bovine sialoglycoproteins or raw milk proteins on the viability of NHDF cells.(C) Morphological appearances of NHDF cells cultured in serum-free DMEM with or without bovine sialoglycoproteins.Scale bar = 200 μm.(D) Doubling time of NHDF cells cultured in serum-free DMEM with or without bovine sialoglycoproteins.The data were obtained from three replicates.*p < 0.05, **p < 0.01, ***p < 0.001.
5 mg/mL of bovine sialoglycoproteins treatment was able to promote the cell scratch healing more rapidly compared with the control.Especially at 36, 48, and 60 h of treatment, there was a significantly difference in wound closure rate (%) between the treated and control groups, which was 27.2 versus 21.2 (p < 0.01), 31.7 versus 25.1 (p < 0.001) and 34.8 versus 27.9 dicated that bovine sialoglycoproteins have a potential ability to accelerate skin wound healing.F I G U R E 2 Effect of bovine sialoglycoproteins on scratch wound assay.(A) Images of NHDF cells cultured in serum-free DMEM with or without 0.5 mg/mL bovine sialoglycoproteins at different times after scratching.(B) The percentage of wound closure by bovine sialoglycoproteins treated and untreated NHDF cells.Scale bar = 200 μm.The data were obtained from three replicates.Ctrl: control, Sia-P: bovine sialoglycoproteins, *p < 0.05, **p < 0.01, ***p < 0.001.

F I G U R E 3 3 . 5 |
Bovine sialoglycoproteins accelerated the contraction of FPCL.(A) Images of FPCLs at different times of incubation in serum-free DMEM with or without 0.5 mg/mL bovine sialoglycoproteins.(B) Morphological appearances of NHDF cells in PFCLs.Scale bar = 200 μm.(C) The contraction rate (%) of FPCLs treated and untreated with bovine sialoglycoproteins.The data were obtained from three replicates.Ctrl: control, Sia-P: bovine sialoglycoproteins, *p < 0.05, **p < 0.01.secretion of COL-I and TGF-β1 but stimulate the production of FGF-2 in NHDF cells.Bovine sialoglycoproteins increased α 2,6-sialylation on NHDF cell surfaces To verify the effects of bovine sialoglycoproteins on the sialylation of NHDF cells, Cy5 fluorescent dye labeled MAL-II (specificity for α2,3-linked sialic acids) and SNA (specificity for α2,6-linked sialic acids) were used to visualize the sialic acids in NHDF cells.The results showed that sialic acids in NHDF cells were mainly distributed on the cell surface, and bovine sialoglycoproteins treatment could dramatically enhance the α2,6-sialylation but not α2,3-sialylation on the cell surface (Figure 5A, B).In addition, sliver nitrate staining results showed that the protein components of both samples were similar except for a few protein bands with molecular weights of around 70 and 25 kDa, which were abundant in treated NHDF cells.Lectin blotting results showed that the protein bands in treated NHDF cell sample bound to MAL-II slightly stronger than those in untreated NHDF cells sample, whereas the binding capacity of treated NHDF cell samples to SNA was significantly increased (Figure 5C).In support of these observations, RT-qPCR and western blot results showed that the expression of the α2,6-sialyltransferase I (ST6GAL1), which transfer sialic acid to galactose residues of glycans with a α2,6-linkage, was increased in the treated NHDF cells (Figure 5D, E).These results indicated that bovine sialoglycoproteins treatment was able to enhance the α-2,6-sialylation on NHDF cell surfaces concordant with an increase in ST6Gal1 expression.

F I G U R E 4
Therefore, improvement of the cell viability of fibroblasts may delay skin aging.The present study demonstrated that bovine sialoglycoproteins improved the cell viability and shortened the doubling time of NHDF cells, indicating their potential application against skin aging.Bovine sialoglycoproteins for the effects of COL-I, TGF-β1 and FGF-2 secreted by NHDF cells.(A) Gene expression levels of COL-I, TGF-β1 and FGF-2 in NHDF cells were detected by RT-qPCR.(B) Protein expression levels of COL-I, TGF-β1, and FGF-2 secreted by NHDF cells were measured by ELISA assays.The data were obtained from three replicates.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

F I G U R E 5
Effect of bovine sialoglycoproteins on the sialylation of NHDF cells.(A) Lectin histopathologic examination of Cy5 labeled MAL-II and SNA staining performed on NHDF cells.Scale bar = 50 μm, (B) Average fluorescence intensity of Cy5-labeled MAL-II or SNA bound on NHDF cells.(C) Silver nitrate staining and lectin blotting assays of the cell membrane proteins.(D) Gene expression level of ST6GAL1 detected by RT-qPCR.(E) Protein expression level of ST6GAL1 detected by western-blot.The data were acquired from three replicates.Ctrl: control, Sia-P: bovine sialoglycoproteins, *p < 0.05, **p < 0.01, ***p < 0.001.

Fortunately, bovine sialoglycoproteins
enhanced the proliferation and migration of NHDF cells, and decreased the expression of TGF-β1 and COL-I.FGF-2 is another multipotent cytokine that regulates cell growth as well as differentiation, matrix composition, and migration in numerous cell types.It is known that FGF-2 stimulates proliferation of cultured fibroblasts via the ERK1/2 and JNK pathways and promotes fibroblasts migration in wound healing by regulating PI3-Kinase-Rac-JNK and NF-κB-JNKs pathway.11,29,30FGF-2 could also suppress COL-I accumulation and scar tissue formation during wound healing.12,31Furthermore,