Seawater pearl hydrolysate inhibits photoaging via decreasing oxidative stress, autophagy and apoptosis of Ultraviolet B‐induced human skin keratinocytes

Ultraviolet (UV) is the main reason to cause photoaging skin which not only hinders beauty, brings the patients with psychological burden, but also pathologically leads to the occurrence of tumors in skin.


| INTRODUC TI ON
Many factors in the environment, such as ultraviolet (UV) light, chemicals, smoke, etc., can cause photoaging, among which UV light is the most important reason as it can trigger a sequence of cellular reactions and molecular changes in the skin, and ultimately lead to rapid dynamic disorder.Clinically, symptoms in photoaging skin occur with hyperplasia of epidermal cells, followed by atrophy, wrinkling, loose nodes, telangiectasia, disordered chromatosis and coarse pores of skin. 1 Beside damaging people's looks, what is more, photoaging in skin also brings mental burden to patients, weakens self-confidence, affects interaction in social occasion.In addition, it pathologically leads to the occurrence of tumors in skin, which draws wide attention. 2 recent years, the treatment methods of skin photoaging developed rapidly, and the photophysical treatment methods such as laser and intense pulse light play a vital role in treating skin photoaging.Although its efficacy on treating skin aging is significant, it cannot be used in daily skin care supplies to guard against occurrence of skin photoaging among the public.What kind of drugs, especially natural products with few side effects, shall be selected to prevent and block photoaging, so that they can be widely used and accepted in clinical practice, and truly improve the skin health of the public needs further research, which is also the direction of future breakthroughs. 3pu pearl, also called seawater pearl, has been long used as an effective composition in Chinese medicine and pass down to modern times ever since.As stated in the Pharmacopeia of the People's Republic of China, it has good effects in detoxication and tissue regeneration, soothing nerves, eye care and pannus removal.Ancient master Shizhen Li holds that pearls had a cosmetic effect on people's body.5][6] In the early stage, our research group has discovered that seawater pearl powder works well in prevention and treatment of skin photoaging in mice. 7,8arls are insoluble in water.When taken orally or externally, the effective ingredients that can be absorbed by the human body are very limited.Whereas the hydrolyzed pearl solution, after processed by the low-temperature hydrolysis technology, not only preserves the whole ingredients of the pearl, but also enables the active and effective ingredients to be absorbed by the human body to the maximum extent, giving full play to the physiological effects of the pearl, which is suitable for clinical application.Earlier, through hydrolysis of seawater pearls, our research group acquired a variety of products, including hydrolyzed seawater pearl powder, seawater pearl hydrolysate (SPH), Zhenmi tablets and succeeded to develop a novel pearl

| Materials
Seawater pearl hydrolysate (production approval number:

| Main reagents and instruments
Copanlisib was purchased from Aimeijie Technology Co., Ltd.

| Effect determination of SPH on the Hacat cells proliferation with CCK8 method
Measure out 60 mL's SPH with concentration of 1 g L −1 , and add to conventional Hacat cell culture media to obtain 100 mL's volume.
Prepare an array of 600 mg L −1 mother solution, then sterilize them through a microfiltration membrane, and add conventional Hacat cell culture media to dilute it to 50, 100 and 200 mg L −1 .Hacat cells were cultivated using a conventional culture media consisting of DMEM medium with high glucose, 1% P/S antibiotics and 10% fetal cow serum.Cells were cultured at condition of 5% CO 2 and temperature 37°C in the incubator.The medium was renewed per 48 h.Hacat cells in log phase growth at 1 × 10 5 cells mL −1 was digested by trypsin to prepare single cell suspension, which was then added to 96-well culture plate for 48-h culture.After being absorbed, the culture media was discarded.Fresh PBS was added to all wells to flush cells.Then PBS was discarded, and the cells were administered with 50, 100, 200 mg L −1 SPH media and conventional media respectively, and cultured for 24 and 48 h.A 10 μL CCK-8 liquid was added to these wells, followed by another 2-h incubation.
The absorbance value at 450 nm was determined with an enzyme labeling device.The conventional media worked as the control group.
The blank well worked as the blank group.The cell proliferation rate % = (experimental group − blank group)/(control group − blank group) × 100%.

| Effect measurement of UVB irradiation dose on the Hacat cells proliferation using CCK8 method
In this experiment, Hacat cells in log phase growth at 1 × 10 5 cells mL −1 was digested by trypsin, to prepare a single cell suspension, which was then added to six-well plates for 24-h culture.
After being absorbed, the culture media was discarded.Fresh PBS was added to all wells to flush cells.The plates were exposed to radiation by a UVB/UV lighting unit, and the unit was regulated respectively to met the requirements on irradiation intensity of 0/25/50/100/200/400 mJ cm −2 .The irradiation intensity of 0 J cm −2 worked as the control group.After continuous cultivation for 24 h, partial cells were transferred to a 96-well culture plate, and a CCK-8 liquid of 10 μL was added to all these wells, followed by another 2-h incubation.The absorbance value at 450 nm was determined with an enzyme labeling device.The blank well worked as the blank group.
The inhibition rate was calculated based on the equation below to obtain the optimal intensity of UVB irradiation.The cell inhibition rate % = (control group − experimental group)/(control group − blank group) × 100%. 9

| The first time: Grouping and administration of Hacat cells
In this experiment, Hacat cells in log phase growth at 1 × 10 5 cells mL −1 was digested by trypsin, to prepare a single cell suspension, which was then added to six-well plates for 24-h culture.
After being absorbed, the culture media was discarded.Fresh PBS was added to all wells to flush cells.The cells were distributed into seven groups, including normal (N) group, UVB-induced group (UVB), low-dose SPH group (SPHL), medium-dose SPH group (SPHM) and high-dose SPH group (SPHH).All groups but the N group were irradiated under a UVB/UV lighting unit, and the unit was regulated until the irradiation intensity reached 200 mJ cm −2 .
Then discard the PBS and add conventional media to the N group and UVB group.The SPHL, SPHM, and SPHH groups were administrated with 50, 100, and 200 mg L −1 SPH media respectively for 48 h.

| Application of flow cytometry in ROS measurement in Hacat cells
After being cultivated in six-well plates for 24 and 48-h for each group, the cells were digested with trypsin, transferred in 1.5-mL centrifuge tubes, centrifuged at 300 g for 5 min, and the supernatant fluid was eliminated.And 0.5 mL of serum-free media was added to all tubes to allow resuspension for the cells, and then 0.5 μL of DCFH-DA staining fluid was added.The samples were placed upside down to allow homogeneous mixing.The cells were cultivated at condition of temperature 37°C for 20 min, and placed upside down to mix per 5 min.After cultivation at 37°C, the samples underwent centrifugation for 3 min at 300 g and 4°C, the cells settled, and the supernatant fluid was eliminated.The cells were flushed with a serum-free media for three times, underwent centrifugation for 3 min at 300 g and 4°C, precipitated, and the supernatant fluid was eliminated.After resuspension for the cells in a serum-free media, a flow cytometry analysis was conducted, with excitation wavelength of 488 nm and emission wavelength of 525 nm.The ROS level was measured. 10

| Biochemical measurement on levels of MDA and activities of SOD, CAT and GSH-PX in Hacat cells
The cells in all groups were cultivated in six-well plates for 24 and 48 h, digested with trypsin, transferred to 1.5 mL centrifuge tubes, dissolved, underwent centrifugation for 10 min at 4°C and 2000 g, and the supernatant fluid was taken for analysis.Following the biochemical Kit' guidelines, indexes including the contents of MDA, activities of SOD, CAT, and GSH-PX in the cells of each group were measured.The values were expressed in relative units per mg of soluble protein.

| Enzyme linked immunosorbent assay for levels of PC and NT in Hacat cells
The cells in all groups were cultivated in six-well plates for 24 and 48 h, digested with trypsin, transferred to 1.5 mL centrifuge tubes, dissolved, underwent centrifugation for 10 min at 4°C and 2000 g, and the supernatant fluid was taken for analysis.Using the ELISA Kit' guidelines, the levels of PC and NT in the cells of each group were measured.The values were expressed in relative units per mg of soluble protein.

| Detection of aging level of Hacat cells with β-galactosidase staining
After 24 and 48 h of cultivation in six-well plates, the cells in all groups were flushed twice with PBS liquid, and at room temperature a β-galactosidase staining fixative was used for 15 min.The samples were flushed thrice with a PBS liquid, a proper amount of β-galactosidase staining operating fluid was added to all holes, and the samples underwent overnight a water bath at 37°C.The cells were flushed twice with PBS liquid, and the aging level of the cells was observed through a general optical microscope.The aging cells were seen with color of dark blue.The image-pro plus software was adopted to determine the average luminous density of the βgalactosidase positive areas. 11

| Flow cytometry detection of apoptosis level of Hacat cells
The cells in all groups were cultivated in six-well plates for 24 and 48 h, digested with trypsin, transferred to 1.5 mL centrifuge tubes, and underwent centrifugation for 5 min at 300 g.Following this, the supernatant fluid was eliminated and 1 mL PBS was added to all tubes.The cells underwent resuspension and centrifugation for 5 min at 300 g and 4°C, and the supernatant fluid was eliminated.
The precipitated material was resuspended by the added 200 μL pre-cooled binding buffer.And 5 μL Annexin V-FITC was added to mix well with the samples, which was then cultivated in the dark for 10 min.Then, 5 μL PI was added to mix well with the samples, which was then cultivated in the dark for another 5 min.Subsequently, another 200 μL pre-cooled binding fluid was added.After blending, flow cytometry analysis was conducted to detect the level of apoptosis in all groups. 12

| The second time: Grouping and administration of Hacat cells
In this experiment, Hacat cells in log phase growth at 1 × 10 5 cells mL −1 was digested by trypsin, to prepare a single cell suspension, which was then added to six-well plates for 24-h culture.After being absorbed, the culture media was discarded.

| Construction and identification of AMPK overexpression plasmid
The gene synthesizes the ORF region of AMPK, and then amplifies it with the primer containing Kpn I and XhoI restriction sites (upstream primer: 5′-GTTTA AAC TTA AGC TTG GTACCAT.GCGCA GAC TCA GTT CCT GGA GAAAGAT-3′, downstream primer: 5′-AAACG GGC CCT CTA GAC TCG AGT TAT TGT GCA AGA ATT TTAATTAGATTTG-3′) to obtain the product containing the restriction sites, and then combines the product with the pcDNA3.1 vector containing the same sticky end digested by Kpn I and XhoI enzymes, and uses T4 ligase for binding, Then the coated plate was transformed, the monoclonal strains were amplified and screened by PCR, and the positive clones were sequenced and verified, finally the overexpression plasmid pcDNA-AMPK was obtained. 13

| Cell transfection and AMPK mRNA detection
Hacat cells were inoculated into six-well plates, and routinely cultured until 80% of the cells grew to 80% fusion.The original culture media was discarded, and the cells were randomly distributed into normal control group, pcDNA group and pcDNA-AMPK group.The conventional media was added to normal control group to continue the culture; In the pcDNA group, the medium containing pcDNA (empty-vector plasmid without carrying AMPK) was added for transfection; In pcDNA-AMPK group, the medium containing pcDNA-AMPK was added for transfection.After 24 h of transfection, the expression of AMPK mRNA in cells of each group was detected by qRT-PCR method.and the supernatant liquid, that is, the whole protein solution, was taken.After preparation of separation gel and concentrated gel, the protein samples were added into the sampling aperture.After preparation of methanol activated PVDF membrane and a transfer membrane filter paper, current with a constant rate of 300 mA was passed through the membrane.The transformed membrane was put into the blocking liquid and kept at room temperature for 1 h.The sealing solution was eliminated, and the monoclonal antibodies including anti-PI3K, anti-p-PI3K, anti-p-AMPK, anti-AMPK, anti-Akt, anti-p-Akt, anti-p-mTOR, anti-mTOR, anti-LC3II, anti-LC3I, anti-GAPDH were diluted with a proper diluent and cultivated at 4°C for whole night.The diluted monoclonal antibodies were retrieved and flushed for 5 min per time with TBST three times.After the diluted goat anti-rabbit IgG-HRP secondary antibody was added, samples were cultivated for 30 min at room temperature.The samples were washed with TBST four times for 5 min per time on a vibrating table at room temperature.Freshly blended ECL solution was transferred to the membrane's protein side, which was to be exposed in the dark.The photographic film was stored, scanned, and analyzed. 14

| Detection of autophagy level of Hacat cells by immunofluorescence staining
After cultivation for 48 h in six-well plates, pour out the cell culture medium of each group and wash it with PBS three times for 5 min per time.Add 4% paraformaldehyde and fix it for 10 min.After washing with PBS buffer solution, add 5% goat serum and seal it at room temperature for 1 h.After discarding the serum, wash it with PBS buffer solution for three times, and then add rabbit anti human LC3II antibody (1:100) for treatment as above.The first antibody was discarded the next day.After the PBS buffer was fully rinsed, FITC labeled goat anti rat fluorescent second antibody was added and incubated for 1 h in a 37°C incubator under dark conditions.Discard the secondary antibody in the dark room, fully flush with PBS buffer solution, and add DAPI for staining for 5 min.The DAPI staining solution was discarded, the cells were washed with PBS buffer solution and resuspended, and the images were carefully observed and scanned with a confocal microscope under dark conditions. 15

| Statistical analysis
The results are presented as the mean ± standard error (SE), and SPSS (version 17.0) (IBM) software was employed to conduct statistical analysis.The least significant difference (LSD) test and onefactor analysis of variance (ANOVA) were used to determine the differences among all groups; p < 0.05 was viewed as significant.All the mathematical or statistical graphics were output from origin 8.6 software (OriginLab).

| Effect of SPH on the proliferation of Hacat cells
In comparison with the control group, the dose with 200 mg L −1 SPH significantly raised speed of cell proliferation after 24 and 48 h of cultivation (p < 0.05), by contrast, neither dose with 50 nor 100 mg L −1 SPH showed significant difference after 24 and 48 h of cultivation (p > 0.05) (Figure 1).Therefore, the proliferation of Hacat cells was promoted by SPH without toxicity.

| Effect of UVB irradiation intensity on the proliferation of Hacat cells
The cell growth inhibition rate increased as UVB irradiation intensity rose, which shows intensity-dependent pattern within a certain intensity range.The half inhibitory dose was 200 mJ cm −2 (Figure 2), which proved to be the best UVB irradiation intensity to be used in the follow-up experiment.

F I G U R E 1
Effect of SPH on the cell growth inhibition rate of Hacat cells after 24 and 48 h of cultivation.Different letters indicate that the difference is significant between two groups (p < 0.05); error bar = SE (n = 3).In comparison with the N group, the activities of SOD and GSH-PX in Hacat cells in the UVB, SPHL, SPHM and SPHH groups declined significantly after cultivation for 24 and 48 h (p < 0.05), the activity of CAT in Hacat cells in the UVB, SPHL, SPHM and SPHH groups declined significantly after cultivation for 24 h (p < 0.05), the activity of CAT in Hacat cells in the UVB, SPHL and SPHM groups decreased significantly after cultivation for 48 h (p < 0.05), but no significant difference was found in the CAT activity in the SPHH group after cultivation for 48 h (p > 0.05) (Figure 4).and in a dose-dependent pattern within a specific concentration range, the activities of SOD, CAT, and GSH-PX increased as SPH rose (Figure 4).

| Effect of SPH on the contents of PC and NT of Hacat cells
In comparison with the N group, the contents of PC and NT in Hacat cells in the UVB, SPHL, SPHM and SPHH groups went up significantly after cultivation for 24 h (p < 0.05), the contents of PC and NT in Hacat cells in the UVB, SPHL and SPHM groups went up significantly after cultivation for 48 h (p < 0.05), but no significant change was shown in the contents of PC and NT in the SPHH group after cultivation for 48 h (p > 0.05) (Figure 5).Compared with the UVB group, the levels of PC and NT in Hacat cells in the SPHL, SPHM and SPHH groups dropped down significantly after cultivation for 24 and 48 h (p < 0.05), and in a dose-dependent pattern within a specific concentration range, the contents of PC and NT decreased as SPH rose (Figure 5).

| Effect of SPH on the aging of Hacat cells
In comparison with the N group, the aging level of Hacat cells in the UVB, SPHL, SPHM and SPHH groups went up significantly after cultivation for 24 and 48 h (p < 0.05) (Figure 6A,B).In comparison with the UVB group, the aging level of Hacat cells in the SPHL, SPHM and SPHH groups dropped down significantly after cultivation for 24 and 48 h (p < 0.05), and in a dose-dependent pattern within a specific concentration range, the aging level decreased as SPH rose (Figure 6A,B).

F I G U R E 4
Effect of SPH on the level of malondialdehyde and activities of SOD, catalase and glutathione peroxidase in Hacat cells after 24 and 48 h of culture.Different letters indicate that the difference is significant between the two groups (p < 0.05); error bar = SE (n = 3).

| Effect of SPH on apoptosis of Hacat cells
In comparison with the N group, the apoptosis index of Hacat cells in the UVB, SPHL, SPHM and SPHH groups went up significantly after cultivation for 24 and 48 h (p < 0.05) (Figure 7A,B).In comparison with the UVB group, the apoptosis index of Hacat cells in the SPHL, SPHM and SPHH groups dropped down significantly after cultivation for 24 and 48 h (p < 0.05), and in a dose-dependent pattern within a specific concentration range, the apoptosis index decreased as SPH rose (Figure 7A,B).
In comparison with the UVB group, the relative expression levels of p-Akt/Akt, p-PI3K/PI3K and p-mTOR/mTOR protein in the SPHH group significantly increased after cultivation for 48 h (p < 0.05), the relative expression levels of p-PI3K/PI3K and p-Akt/Akt protein in the PI3KI, AMPKO and PI3KI + SPHH groups significantly decreased after cultivation for 48 h (p < 0.05), the relative expression levels of p-mTOR/mTOR protein in the PI3KI, AMPKO and AMPKO + SPHH groups significantly declined after cultivation for 48 h (p < 0.05), and the relative expression level of p-mTOR/mTOR protein in the PI3KI + SPHH group significantly increased after cultivation for 48 h (p < 0.05) (Figure 8A,B).
In comparison with the UVB group, the relative expression of p-AMPK/AMPK, LC3II/I and LC3II/GAPDH protein in the SPHH group significantly declined after cultivation for 48 h (p < 0.05), the relative expression level of p-AMPK/AMPK protein in the PI3KI, AMPKO and AMPKO + SPHH groups significantly increased after cultivation for 48 h (p < 0.05), the relative expression level of p-AMPK/AMPK protein in the PI3KI + SPHH group significantly declined after cultivation for 48 h (p < 0.05), the relative expression of LC3II/I and LC3II/GAPDH protein in the PI3KI, AMPKO and PI3KI + SPHH groups significantly increased after cultivation for 48 h (p < 0.05) (Figure 8A,B).
In comparison with the PI3KI group, the relative expression level of p-Akt/Akt and p-mTOR/mTOR protein in the PI3KI + SPHH group significantly increased after cultivation for 48 h (p < 0.05), the relative expression of p-AMPK/AMPK, LC3II/I and LC3II/GAPDH protein in the PI3KI + SPHH group significantly decreased after cultivation for 48 h (p < 0.05) (Figure 8A,B).In comparison with the AMPKO group, the relative expression levels of p-PI3K/PI3K, p-Akt/ Akt and p-mTOR/mTOR protein in the AMPKO + SPHH group were significantly increased after cultivation for 48 h (p < 0.05), the relative expression levels of p-AMPK/AMPK, LC3II/I and LC3II/GAPDH protein in the AMPKO + SPHH group significantly declined after cultivation for 48 h (p < 0.05) (Figure 8A,B).

| Effect of SPH on the autophagy level of Hacat cells
The distribution of autophagy specific protein LC3II marked by green fluorescent FITC in Hacat cells was clearly seen under laser confocal microscope.In comparison with the N group, the autophagy F I G U R E 5 Effect of SPH on the levels of protein carbonyl compound and nitrosylated tyrosine protein in Hacat cells after 24 and 48 h of culture.Different letters indicate that the difference is significant between the two groups (p < 0.05); error bar = SE (n = 3).The earth's atmosphere can filter out UVC when the sunshine passes through, and both UVA and UVB can induce the skin to produce excessive ROS. 16Excessive ROS causes oxidative stress and brings "oxidative damage" to certain cell components, for example, protein, lipid membrane, mitochondria and DNA, etc.Then, these damages pile up in cells and promote the increase of ROS level, thus resulting in a vicious cycle, that is, reactive oxygen to mitochondrial damage and in turn to increase of reactive oxygen, which is mutual causality.The continuous aggravation of cell damage facilitates the change of cellular signal transduction pathway, matrix metalloproteinases playing their roles, inflammatory reaction and apoptosis, which cause visible skin aging. 17,18Therefore, under the condition that the skin is irradiated by UV in sunlight, the elimination of excessive ROS and oxidative damage of skin cells is the fundamental way to control photoaging. 19ntemporary scholars have proved the high antioxidant capacity of pearl extract in vitro and in vivo, and the effect to prevent and cure sickness caused by ROS. 20,21Previous research found that skin photoaging in mice can be well prevented and cured through using seawater pearl powder, and its effect mechanism involves eliminating excessive ROS from photoaging skin. 7,8Pearl extract can also diminish inflammation and apoptosis of human skin keratinocytes suffering from UVB irradiation, 22 and Pearl water extract can lower down the aging quantity of human skin fibroblasts suffering from UVB irradiation. 23In addition, pearl extract promotes skin damage repair by stimulating the proliferation of human skin fibroblasts, enhancing the migration ability of cells and collagen aggregation. 24,25 current study, the photoaging model of Hacat cells was also established by UVB irradiation.The effect was found to be dose-  However, autophagy also has causal relationship with type-II programmed cell death (PCD); As a double-edged sword, autophagy play the dual role of "promoting survival" and "promoting death" of cells. 31,32In most cases, ROS-activated autophagy under oxidative stress acts as a negative feedback mechanism of cell protection to prevent oxidative damage.However, high-intensity oxidative stress can also inhibit autophagy or trigger excessive autophagy to promote the production of oxidative stress, both of which may cause cell damage or death. 33ring the process of autophagy formation, ROS mainly regulates autophagy by suppressing the activity of Atg4.ROS inactivates Atg4 and causes LC3II accumulation, which increases autophagy. 34erefore, LC3II is a marker protein closely related to autophagy. 35 present, studies have found that low dose 1. to the significant skin protection capabilities, SPH shows great potential to be applied in the development of skin care products.
hydrolysis technology (national invention patents zl95106283.2,zl95108034.2, and zl95107894.1).The wavelength of UVB is mostly absorbed by the protein and DNA in the keratinocytes of the epidermis of the human skin.At the same dose, the damage intensity of UVB to skin was about 1000 times that of UVA.Therefore, this study intends to use the UVB-induced Hacat cell photoaging model of human skin keratinocytes to measure the changes of cell proliferation, oxidative stress, aging, apoptosis, autophagy and autophagy-related protein and signal pathway expression before and after the intervention of SPH, and to further go into the molecular biological mechanism of SPH in preventing and treating photoaging together with the intervention outcome of Copanlisib (PI3K inhibitor) and recombinant plasmid pcDNA3.1(+)/AMPK(AMPK overexpression).
450522020014) was donated by Beihai Baozhulin Marine Technology Co., Ltd; Hacat cells were bought from the Kunming Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Sciences.

Fresh
PBS was added to all wells to flush cells.The cells were distributed into seven groups, including normal (N) group, UVBinduced group (UVB), high-dose SPH group (SPHH), PI3K inhibition group (PI3KI), AMPK overexpression group (AMPKO), PI3K inhibition + high-dose SPH group (PI3KI + SPHH), AMPK overexpression + high-dose SPH group (AMPKO + SPHH).All groups but the N group were irradiated under a UVB/UV lighting unit, and the unit was regulated until the irradiation intensity reached 200 mJ cm −2 .Then discard the PBS and add conventional media to the N group and UVB group.The SPHH groups were administrated with 200 mg L −1 SPH media, and the PI3KI group was treated with 0.2 μmol L −1 copanlisib media for 48 h.The cells in AMPKO group were transfected with pcDNA3.1 (+)/AMPK and then added conventional media for 48 h.The PI3KI+ SPHH group were treated with 200 mg L −1 SPH + 0.2 μmol/L copanlisib media for 48 h.The cells in AMPKO + SPHH group were transfected with pcDNA3.1 (+)/AMPK and then added 200 mg L −1 SPH media for 48 h.

2. 6 . 3 |
Western blot test for the expression of autophagy associated proteins and signaling pathways in Hacat cells After cultivation for 48 h in six-well plates, the cells in all groups were flushed twice with PBS liquid, and the remaining cells were dried as wholly as possible.The cells were dissolved through adding a proper amount of total cell protein extraction reagent for 3-5 min.The cells and reagents were transferred into a 1.5 mL centrifuge tube, immersed in ice cold water for 30 min, and blown with a pipette repeatedly to ensure cell dissolution wholly.The samples underwent centrifugation for 5 min at 13 000 g and 4°C,

3 |
Effect of SPH on the ROS level of Hacat cellsIn comparison with the N group, the ROS level of Hacat cells in the UVB, SPHL, SPHM and SPHH groups went up significantly after 24 and 48 h of cultivation (p < 0.05) (Figure3A,B).In comparison with the UVB group, the ROS level of Hacat cells in the SPHL, SPHM and SPHH groups went down significantly after 24 and 48 h of cultivation (p < 0.05), and in a dose-dependent pattern, the ROS level decreased as SPH rose within a specific concentration range (Figure3A,B).

3. 4 |
Effect of SPH on the levels of MDA and activities of SOD, CAT, and GSH-PX in Hacat cells In comparison with the N group, the MDA level of Hacat cells in the UVB, SPHL, SPHM and SPHH groups went up significantly after 24 h of cultivation (p < 0.05), the MDA level of Hacat cells in the UVB, SPHL and SPHM groups rose significantly after cultivation for 48 h (p < 0.05), but no significant change was shown in the MDA level in the SPHH group after 48 h of cultivation (p > 0.05).Compared with the UVB group, the MDA level of the Hacat cells in the SPHL, SPHM and SPHH groups dropped significantly after cultivation for 24 and 48 h (p < 0.05), and in a dose-dependent pattern within a specific concentration range, the MDA content decreased as SPH rose (Figure 4).
Compared with the UVB group, the activities of SOD, CAT, and GSH-PX in the Hacat cells in the SPHL, SPHM and SPHH groups went up significantly after cultivation for 24 and 48 h (p < 0.05), F I G U R E 2 Effect of UVB irradiation intensity on the cell growth inhibition rate of Hacat cells after 24 h of cultivation; error bar = SE (n = 3).

F
I G U R E 3 (A) Effect of SPH on reactive oxygen species (ROS) level of Hacat cells detected by flow cytometry after 24 and 48 h.(B) Effect of SPH on ROS level of Hacat cells after 24 and 48 h.Different letters indicate that the difference is significant between the two groups (p < 0.05); error bar = SE (n = 3).
cells in the UVB, PI3KI, AMPKO, PI3KI + SPHH and AMPKO + SPHH groups went up significantly after cultivation for 48 h (p < 0.05), but no significant change was shown in the autophagy level of the SPHH group (p > 0.05) (Figure 9A,B).In comparison with the UVB group, the autophagy level of Hacat cells in the SPHH group declined significantly after cultivation for 48 h (p < 0.05), and the autophagy level of Hacat cells in the PI3KI, AMPKO and PI3KI + SPHH groups increased significantly after cultivation for 48 h (p < 0.05) (Figure 9A,B).Compared with the PI3KI group, the autophagy level of Hacat cells in the PI3KI + SPHH group declined significantly after cultivation for 48 h (p < 0.05) (Figure 9A,B).In comparison with the AMPKO group, the autophagy level of Hacat cells in the AMPKO + SPHH group dropped down significantly after cultivation for 48 h (p < 0.05) (Figure 9A,B).

F
I G U R E 6 (A) Effect of SPH on the aging level of Hacat cells detected by β-galactosidase staining after 24 and 48 h of culture (×200), scale bar = 50 μm.(B) Effect of SPH on the aging level of Hacat cells after 24 and 48 h of culture.Different letters indicate that the difference is significant between two groups (p < 0.05); error bar = SE (n = 3).
7 (A) Effect of seawater pearl hydrolysate (SPH) on the apoptosis level of Hacat cells was measured by flow cytometry after 24 and 48 h of culture.(B) Effect of SPH on the apoptosis level of Hacat cells after 24 and 48 h of culture.Different letters indicate that the difference is significant between two groups (p < 0.05); error bar = SE (n = 3).
U R E 8 (A) Effect of SPH on the relative expression levels of p-PI3K, PI3K, p-Akt, Akt, p-AMPK, AMPK, p-mTOR, mTOR, LC3, GAPDH protein measured by Western blot in Hacat cells after cultivation for 48 h.(B) Effect of SPH on the relative expressions of autophagy associated proteins and signaling pathways in Hacat cells after cultivation for 48 h.Semi-quantitative analysis based on the relative density of p-PI3K, PI3K, p-Akt, Akt, p-AMPK, AMPK, p-mTOR, mTOR, LC3II, LC3I.Data were normalized with GAPDH as the internal reference.Different letters indicate that the difference is significant between the two groups (p < 0.05); error bar = SE (n = 3).
In vivo environments, normally CAT, SOD and GSH-PX are functional enzymes that can eliminate ROS off the body, keep the balance of oxidation and antioxidant systems dynamically, and guard skin tissue against oxidative damage. 26,27Too much ROS can impair the enzymatic antioxidant system in skin cells, which destroys the dynamic balance between oxidation and antioxidant system in human body, damaging the function of the body to eliminate ROS, causing increased ROS accumulation in vivo.Increased ROS accumulation is harmful to liposomes and protein in skin cells, which leads to cell apoptosis or necrosis.Piao et al. 28 reported that UVB irradiation of Hacat cells can enhance the ROS level, lipid peroxidation 8-isoprostane, protein carbonylation and the apoptosis index.Wang et al. 29 reported that the UVB irradiation of Hacat cells can significantly lower down the activities of endocellular SOD and GSH-PX, enhance the ROS level, lipid peroxidation MDA and apoptosis index, indicating that photoaging Hacat cells underwent oxidative stress.The state of oxidative stress was further verified by the UVB group of photoaging Hacat cells in current study.After UVB irradiation, the ROS, MDA, protein carbonylation PC and NT contents, apoptosis rate and aging level of photoaging Hacat cells increased, accompanied by the decline of the CAT, SOD and GSH-PX activities of photoaging Hacat cells.SPH treatment eased oxidative stress, reduced apoptosis index and aging level through increasing the CAT, SOD and GSH-PX activity of photoaging Hacat cells and reducing the ROS, MDA, PC and NT contents of photoaging Hacat cells.After SPH treatment, a dose-dependent pattern within a certain concentration range were shown in the results, among which the 200 mg L −1 dose performed best.Therefore, by eliminating excessive ROS and relieving oxidative stress, SPH can effectively inhibit apoptosis and aging.Autophagy is a kind of self eating phenomenon commonly existing in eukaryotic cells.It is one of the important ways of cell repair to enable cells to survive under stress by recycling nutrients by degrading long-standing proteins and damaged organelles in cells 30 ;

5 |
photoaging Hacat cells to reduce autophagy level.These data could support that SPH can reduce the autophagy level by clearing the excessive ROS caused by 200 mJ cm −2 UVB irradiation on Hacat cells, reducing AMPK expression, increasing PI3K-Akt expression, and activating mTOR.