A study on the delayed effect of tilapia skin collagen on skin aging for mice and its possible mechanism

Study the possible mechanism and delayed effect of tilapia skin collagen on skin aging for mice.

| 3437 PU and QU dry and rough.At present, the collagen is hydrolyzed into the oligopeptides with small molecular weight and good permeability by enzymatic hydrolysis as raw materials of cosmetics or functional foods.People often supplement collagen to achieve beauty and health of delaying aging. 6,7Fish skin has the advantages of easy access, less pollution, less disease transmission risk, no religious barriers, and high production of collagen, thus becoming a better source of collagen. 8Tilapia is an important freshwater fish species in the world.Over the past few years, tilapia has grown steadily in production and processing, and has become one of the leading exporters for aquatic products.In order to effectively improve the scale application of tilapia skin and confirm its antioxidant, anti-aging activity, the study has proved the delayed effects of tilapia skin collagen on skin aging and its possible mechanism.

| Preparation of aging model
The KM mice were acclimated under the same feeding conditions for 1 week.Before the experiment, depilation was carried out by the hair clipper and depilatory at the back of mice.The model was induced by D-galactose.The mice were injected subcutaneously 5% D-galactose at a daily dose of 1 g/kg.Meanwhile, UVA light was jointly used to establish the aging model.The irradiation wavelength was 350-400 nm, the irradiation intensity was 300 μW/cm 2 , and the irradiation time was 60 min/d.

| Grouping and treatment
Kunming (KM) mice were randomly divided into the aging model group, the normal group, the positive control group (vitamin E) and the low, medium, high dose tilapia skin collagen group (2.0, 4.0, 8.0 mg/g), 25 in each group.The normal group was only injected with saline at the back and the neck.Other groups were established the aging model as above for 50 days.After modeling, the positive control group was treated with a dose of 10% vitamin E once a day, and the low, medium, high dose tilapia skin collagen group was separately applied 2.0, 4.0, 8.0 mg/g of tilapia skin collagen for 40 days.
The changes of skin tissue morphology, water content, Hyp content, and SOD activity in mice were studied at the day 10, 20, 30, 40, 50.

| Sample collection and processing
Five mice were executed in each group at the day 10, 20, 30, 40, 50.
The skin tissues were taken, and some of them were put into 4% paraformaldehyde to soak and fix.After weighing, labeling and recording, others were quickly put into frozen storage tubes and stored in the liquid nitrogen for subsequent experimental needs.

| Observation of skin superficial characteristics
The appearance of skin color, smoothness, and wrinkles was observed on the back of mice.

| Histopathological observation
The hairless skin tissues were fixed with 10% neutral formalin, routinely dehydrated, embedded in paraffin, sectioned.After preparation of samples, HE staining and Masson staining were performed.
The histopathological changes were observed under an optical microscope, such as changes in skin thickness, pore, and hair follicles.
The MShot Image Analysis System was used to calculate the thickness of dermis quantitatively.Masson staining was used to observe the content and distribution of collagen fibers in dermis.

| Determination of moisture content
The stratum corneum hydration (SCH) and the transdermal water loss (TEWL) of the skin tissue were detected by the Cutometer dual MPA 580 skin tester according to instrument description.The detection location was dorsal skin of the mice, and the detection was carried out with the indoor temperature between 20 and 22°C and the ambient humidity between 40% and 60%.

| Statistical analysis of data
The resulting data were analyzed and processed with IBM SPSS 21.0.The comparison between two groups were performed with t-test, and the comparison among multiple groups was performed with one-way ANOVA.The experimental data were expressed as (x ± s).p < 0.05 was considered to be statistically significant.

| Results of superficial characteristics
During the experiment, mice in the normal group had smooth skin, normal color, and no wrinkles.The mice skin in the aging model group gradually became dry, less elastic, relaxed wrinkled, and the color was dark.Mice in the positive control group were significantly better than those in the aging model group, but slightly worse than those in the normal group.Compared with the aging model group, the skin condition of mice in the different dose tilapia skin collagen groups was significantly improved, and that of the high dose tilapia collagen group was better than that in the positive control group and the medium, low dose tilapia skin collagen group.In the high dose tilapia skin collagen group, the drying was alleviated, the skin color was improved, and the wrinkles were significantly reduced (Figure 1).

| Results of histopathological observation
The skin of the back was stained by HE and the morphology was boundaries among the basal layer, the acanthocyte layer, the granular layer, and the cuticle were clear.The dermis was located beneath the epidermis, which contained blood vessels, sweat glands, hair follicles, and sebaceous glands.The growth of skin tissue in the aging model group was impaired.Over time, the dermis was significantly thinner and looser, the number of hair follicle and sebaceous glands decreased obviously.The dermal thickness, the number of hair follicles, and sebaceous glands in the positive control group were also decreased, but there were still significant differences between the positive control group and the aging model group.The high dose tilapia skin collagen group showed normal skin tissue morphology with regular epidermal cell layer arrangement and normal dermal integral structures.The number of hair follicles increased obviously and the dermis is more tightly packed compared with the positive control group.There was no significant difference in the histomorphological characteristics between the medium dose tilapia skin collagen and the positive control group.The low-dose collagen group exhibited loosely arranged dermal cell layers and a decreased number and size of hair follicles and sebaceous glands (Figure 2).Dermal thickness of mice in each group is shown in Table 1.
Compared with the normal group, dermal thickness in the aging group significantly decreased (p < 0.01), and the decrease rate of dermal thickness increased with time, indicating that the aging model was successfully constructed.Compared with aging group, the dermal thickness in positive control group, medium and high dose tilapia skin collagen group was significantly increased (p < 0.01), while that in low dose group was not significantly increased (p > 0.05).These   | 3441 PU and QU

| Results of water content
The moisture content of aging skin is reduced by thinning of the epidermis and dermis.and the higher dose of tilapia skin collagen, the higher water content.These results indicate that tilapia skin collagen can improve skin physiological function and delay skin aging by reducing TEWL and increasing epidermal humidity (Tables 2 and 3).

| Results of Hyp content
Hyp is a major and relatively constant amino acid in fiber and collagen.The degree of skin aging can be determined according to the content of Hyp.With age, the Hyp content in skin decreases, which blocks the synthesis of collagen and also results in reduction of collagen fiber content. 10,11In this study, the Hyp content of each group decreased gradually, the aging model group was the fastest, and the overall level was less than that of the normal group(p < 0.05).
The Hyp levels in all tilapia skin collagen groups and positive control group were higher than that in the aging group(p < 0.05 or p < 0.01), and the higher dose of tilapia skin collagen, the higher Hyp content, the slower rate of decrease in the experimental process (Figure 6, Table 4).

| Results of SOD activity
SOD is an important enzyme in the body with free radicals as substrates which plays the very important role in scavenging free radicals.Its activity is closely related to aging, so SOD activity value can be used as an important reference index for skin aging. 12The results showed that the SOD activity of each group decreased gradually, the aging model group was the fastest, and the overall level was less than that of the normal group (p < 0.01).The SOD activity in all collagen groups and positive control group were higher than that in the aging group (p < 0.01), and the higher dose of tilapia skin collagen, the higher SOD activity, and the slower rate of decrease in the experimental process.It shows that vitamin E and tilapia skin collagen can effectively ease the aging process (Figure 7, Table 5).

| DISCUSS ION
As a superficial organ of the human body, skin can reflect the aging state of whole body, and it is also the most intuitive reference to judge the aging degree of body.Skin aging is a complicated evolution

| CON CLUS ION
The collagen of tilapia can change the thickness of epidermis and dermal, significantly increase the skin moisture content of mice, improve the Hyp content and SOD activity of aging mice.The aging process of mice skin was effectively alleviated, and the aging symptoms of mice were significantly improved.

ACK N OWLED G M ENTS
This work has no financial support.We thank Xuxiaoxiao worked in Slice chamber of Yantai Yuhuangding Hospital for technical supports and substantial helps.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
This study was approved by Ethics Committee in Yantai Yuhuangding Hospital (Approval no.LDSW2022055).

2. 6 . 4 |
Determination of Hyp content 50 mg tissue sample was cut slightly, the absorbance of sample solution was detected according to the instruction of Hyp kit, and the change of Hyp content was compared between the samples.

2. 6 . 5 |
Determination of SOD content 200 mg tissue sample was cut by an eye scissors.Pour into the homogenate tube, add 1.8 mL cold physiological saline to homogenate, thoroughly grind.After getting 10% tissue homogenate, centrifuge for 15 min at 3000 r/min.The SOD content of supernatant was detected by SOD kit.
observed under an optical microscope.The skin form of the normal group remains normal, skin tissue are stained dark red, and F I G U R E 1 Macroscopic observation of mice skin (50d).(A) The normal group.(B) The aging model group.(C) The positive control group.(D) The high-dose collagen group.(E) The medium-dose collagen group.(F) The low-dose collagen group.
results indicated that vitamin E and tilapia skin collagen could delay dermal thinning, and the effect of tilapia skin collagen at high dose was more significant.Collagen fibers are the main component of the dermis, accounting for 95%-98% of the total fiber mass.Changes in dermis structure are the main causes of skin aging, such as decreased collagen fibers and thinning of the dermis.Collagen fibers were F I G U R E 2 Results of histopathological observation (HE staining; magnification, ×100; 50d).(A) The normal group.(B) The aging model group.(C) The positive control group.(D) The high-dose collagen group.(E) The medium-dose collagen group.(F) The low-dose collagen group.TA B L E 1 Effect of tilapia skin collagen dose on dermal thickness (x ± s, n = 5, μm).

F I G U R E 3 F I G U R E 4
stained blue following Masson staining, so it can be used to observe the density and distribution of collagen fibers in the dermis.As shown in Figure3, the collagen fibers of the skin in the normal group were blue-purple with relatively uniform staining, indicating that collagen fibers were abundant.Compared to the normal group, the collagen fibers in the aging model group exhibit light coloration and sparse alignment.The collagen fibers in the high dose tilapia skin collagen group were uniform, dark blue and closely aligned, close to those in the normal group.More abundant collagen fibers were found in the low and medium dose tilapia skin collagen groups compared to the aging group using Masson staining.The results showed that tilapia skin collagen could improve the content and distribution of collagen fibers in the dermis of aging mice, and delay the skin aging of mice.Masson trichrome stain; magnification, ×100.(50d).(A) The normal group.(B) The positive control group.(C) The high-dose collagen group.(D) The medium-dose collagen group.(E) The low-dose collagen group.(F) The aging model group.Results of SCH.

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Transepidermal water loss (TEWL) and SCH are good indexes of skin water content.As shown in Figures 4 and 5, the SCH and TEWL of the normal group remained basically unchanged.SCH decreased and TEWL increased gradually over time in other groups.Compared with the normal group, SCH decreased and TEWL increased significantly in the aging model group with statistical difference (p < 0.01).Compared with the aging model group, SCH increased and TEWL decreased in positive control group and all tilapia skin collagen groups with statistical significance (p < 0.01 or p < 0.05),

F I G U R E 5
Results of TEWL.TA B L E 2 Results of stratum corneum hydration (x ± s, n = 5, %).
process.Factors that affect skin aging include endogenous factors and exogenous factors.Endogenous factors mainly include neuroimmuno-endocrine disorder, hereditary gene, metabolic product damage, etc., and exogenous factors mainly include environmental pollution and sunshine. 13-15At present, natural aging model, Dgalactose induced subacute aging model, photoaging model, and nothymus senescence model are used to study the aging of animals.Among them, the application of D-galactose induced subacute aging model and photoaging model are more.The former is systemic and has no prominent effect on skin aging.D-galactose can result in a series of degenerative changes of cells, leading eventually to organismal aging, such as causing oxidative stress and reducing the ability of cells to clear free radicals, etc.The latter need take a long time, and the histological change is needed for more than 6 months.Aging is the joint result of endogenous physiological aging and exogenous environmental factors.In this study, D-galactose injection was used to cause endogenous aging of skin cells in mice, meanwhile, UVA irradiation was used to cause skin exogenous light injury in mice, which further aggravated the aging of skin.Drying, loose, dark color, wrinkle, the obvious thinning of dermis and reduction of hair follicles, sebaceous glands, water content, Hyp content, and SOD activity have showed that the aging model was successful.Vitamin E is an antioxidant which can scavenge free radicals and has been widely used to delay skin aging.This study compared different doses of tilapia skin collagen groups and positive control group.The results showed that both the positive control group and the collagen groups F I G U R E 6 Results of Hyp content.increased the content of Hyp and SOD activity, and the high-dose collagen was significantly higher than the positive control group and the medium, low dose collagen group.The results showed that tilapia collagen can effectively relieve the aging of skin and achieve anti-aging effect.These possible mechanisms are outlined below.Tilapia skin collagen hydrolysates, such as small molecule oligopeptides, are rich in hydrophobic amino acids, which can be used as raw materials to participate in collagen synthesis and inhibit its degradation, stimulate the proliferation and migration of fibroblasts and the synthesis of hyaluronic acid, and enhance the activity of antioxidant enzymes to alleviate skin oxidative stress, thus achieving the effect of delaying aging.The specific molecular mechanism and pathway can be further discussed in subsequent experiments.[16][17][18]At present, in most anti-aging experiments, tilapia skin collagen is mostly used for oral administration and less for daub.Meanwhile, tilapia skin collagen has good bioactivity, biocompatibility, and permeability, so we can study its application in external beauty products.The anti-aging property of tilapia skin collagen was further demonstrated by studying the effect of different doses and different time on skin aging.