The protects of deep sea water against photoaging of HaCaT keratinocyte induced by UVB via suppression MMPs and MAPKs/NF-κB signaling pathway

doi:10.21203/rs.3.rs-860697/v1

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The protects of deep sea water against photoaging of HaCaT keratinocyte induced by UVB via suppression MMPs and MAPKs/NF-κB signaling pathway

https://doi.org/10.21203/rs.3.rs-860697/v1

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Ultraviolet radiation destroys skin through several harmful effects, like inducing reactive oxygen species, inflammatory response, and collagen degradation. In this study we researched the impact of deep seawater (DSW) on the photoaging of HaCaT keratinocytes induced by ultraviolet B (UVB). DSW can inhibit epidermal hyperplasia and collagen degradation, increase skin moisture and hydroxyproline content, thereby improving the macro and histopathological damage of skin under UVB irradiation. Besides, DSW can inhibit UVB-induced oxidative stress (OS), improve antioxidant enzyme activity and inhibit the cellular signal transduction pathway of inflammatory response. Results showed DSW curbed the UVB irradiated levels of reactive oxygen species, superoxide dismutase (SOD), oxidative enzyme, glutathione peroxidase (GSH-Px), proinflammatory cytokines (TNF-α, IL-6). DSW prevented UVB-induced photoaging by suppressing collagen disorientation and expression of MMPs induced by UVB. Moreover, Western blot analyses exhibited that DSW significantly lessened the protein levels of phosphorylated SAPK/JNK kinase, phosphorylated ERK1/2 kinase, and phosphorylated p38 kinase. Similarly, UVB induced nuclear translocation of nuclear factor kappa-B were diminished by treatment of DSW. Therefore, DSW may lessen skin OS and inflammatory response under UVB irradiation. These data suggest that DSW can be used as a potentially effective skin care and dietary supplement for attenuating UVB-induced premature skin aging.

General Biochemistry

General Cell Biology & Physiology

Immunology

Balanced deep-sea water

Ultraviolet B light

Photoaging

HaCaT keratinocyte

Skin aging is a multi-system degradation course involving skin and skin support systems¹. Exposure to ultraviolet light may lead to oxidative stress (OS), immunosuppression, inflammation, sunburn, nonmelanoma, and premature aging of the skin, and melanoma skin cancer, which is dubbed photoaging, depending on the number and type of ultraviolet exposure and the skin variety of the exposed individual^2–4. In humans, photoaging is characterized by fine and rough wrinkles, dry and rough skin, sun skin, sallow color, poor elasticity recoil, and impaired wound healing^5–7. The current study suggests that the accumulation of ROS reduces the body's salubrity as oxidative damage is a factor leading to aging⁸. However, long-term exposure to ultraviolet radiation can cause severe adverse impacts on skin structure and function, eventually leading to photoaging and even skin cancer⁹.

Aging is a natural course for everyone. It can cause all kinds of diseases. Skin reacts strongly to photoaging resulting from OS caused by reactive oxygen species ¹⁰. Inflammation and its induced inflammation hold the key to the process of human skin photoaging. However, ROS levels are significantly increased under environmental stresses, such as UVB or heat exposure, resulting in significant impairment of cell function and structure. Previous studies¹¹ have exhibited that UV-induced ROS activates lipid mediators, comprising cyclooxygenase-2 (COX-2). In addition, exposure of ultraviolet light UV increases the proinflammatory mediators released by diverse skin cells, leading to the activation of matrix metalloproteinases (MMPs) and matrix-degrading enzymes¹². However, inflammation-activated various matrix degradation matrix proteinases lead to accumulation of non-functional matrix components and abnormal matrix degradation, causing photoaging¹³. Therefore, previous studies^{11, 14} have exhibited MMPs can be employed as the main markers of wrinkles and skin inflammation caused by UVB.

Deep seawater (DSW) constitutes a powerful substance featuring multiple physiological bioactivities¹⁵, like anti-tumor¹⁶, anti-oxidation¹⁷, anti-diabetes^{18, 19} anti-hyperlipidemia^{20, 21} and anti-inflammatory^{22, 23}. Besides, animal experiments and clinical trials exhibited taking DSW can improve atopic eczema^{12, 24}. Furthermore, South Korea, Japan, Taiwan, and the U.S. have also launched cosmetics containing DSW²⁵. Nevertheless, to our knowledge, how DSW affects UVB-induced skin injury hasn’t been completely clarified²⁶. Hence, we evaluated how DSW affects UVB-induced HaCaT keratinocyte injury and further explored the molecular biological regulation mechanism of DSW on UVB-induced inflammation and photoaging of HaCaT cells.

2.1 Chemicals and reagents

SOD, protein extraction kits, catalase ²⁷, glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) came from Nanjing Jiancheng Bioengineering Co., Ltd (Nanjing, China). Anti-fading mounting solution containing 4,6-diamiding-2-phenylindole (DAPI) came from Sigma-Aldrich Chemical Co.(St. Louis, MO, USA.) and alexa 488-binding goat anti-rabbit antibody came from Thermo Fisher Scientific (Waltham, MA, USA). Nitrocellulose filter membrane came from Pall Co. (New York., NY, USA.). Radioimmunoprecipitation assay (RIPA) lysis buffer came from Solarbio Co. (Beijing, China). All other reagents employed in the investigation are analytical grade reagents bought from Kangkede Chemical Reagent Factory (Tianjin, China).

2.2 Fabrication of deep sea water

The intake and the fabrication of DSW were described earlier. For preparing the conditioned medium, dissolve DMEM powder in original hardness DSW (OHDSW, hardness 600, Mg: Ca = 3:1), and sterilize desalted DSW (H0) (hardness 0) and medium by the filtration course. Then, the original hardness DSW was continuously diluted with DSW (H0) for preparing conditioned media containing different hardness levels of DSW (HH(hardness 1800), HM(hardness 1200), and HL(hardness 600)). OHDSW contained 1270 mg/L Mg, 420 mg/L Ca, 309 mg/L Na, and 98 mg/L K. The control and model groups were adjusted employing the same volume of DSW(H0) medium as the DSW (H1800) group. DMEM medium containing synthetic MgSO₄ and CaCl₂ (Mg:Ca = 3:1) was fabricated as Mg + Ca group (hardness 1200) as well.

2.3 Cell treatment and UVB irradiation

HaCaT keratinocyte was provided from Sciences cell bank of Chinese Academy (Shanghai). Routinely culture the cells in DMEM (Gibco) containing 1% antimycotic/antibiotics solution (Gibco) and 10% fetal bovine serum (FBS) (Gibco). For radiating UVB, the cells were rinsed twice employing PBS and stratified employing PBS. PL-S 5W/2P UVB lamp (wavelength 311nm, Philips, Netherlands) was employed for UVB irradiation. The irradiation distance was 10cm (16.7mJ/cm²) and irradiated in the cassette. Cells were cultured in DMEM without FBS for 4h. The UVB intensity at 10cm distance was 0.1mW/cm².

2.4 Cell viability assay

MTT assay was employed for detecting how DSW affects cell viability. After UVB radiation, remove the medium and incubate cells employing DSW with different hardness of H0-H1800 for another 24 hours, and the final volume was 200 µL. Subsequently, alter the medium to 200µL 0.5 mg mL^− 1 MTT solution and incubate them in 5% (v/v) CO₂ at 37°C for 4 hours. Remove the medium and dissolve the precipitate in 100 µL dimethyl sulfoxide for melting the formalin crystal by gently shaking for 10 minutes without light for achieving maximal dissolution. Detect the absorption at OD570nm by a Thermo Scientific Multiskan FC (Waltham, MA, USA). The cell viability was statistically analyzed by analysis of variance, and a T-test was performed by SPSS 25 software (SPSS, Inc. Chicago, II., USA).

2.5 SOD, CAT, GSH-Px, and MDA determination

Plate 5×10⁴ cells per well into a 6-well plate. Cell culture and sample treatment methods refer to above 2.4. Gather the cells, homogenize them employing PBS, and centrifuge them at 2500rpm for 10min at 4℃. Gather the supernatant for evaluating SOD, CAT, and GSH-Px activities and MDA levels, and determined employing the relevant kit on basis of the manufacturer's instructions.

2.6 Immunofluorescence analyses

Immunofluorescence assay was employed for assessing quantitative analyses of ROS production. Cells were incubated on a 6-well plate with a glass slide per plate at the density of 1×10⁵ with DSW (HL, HM, HH) and Mg + Ca, then incubated for 24h and then exposed to UVB. Cell culture pieces were mixed with the working solution at 37°C for 30 minutes, then stained employing ROS staining solution and cultured at 37°C for 30 minutes. Rinse the slides employing PBS solution and stain them employing DAPI for 10 minutes. After washing, immunofluorescence assay was employed for assessing Quantitative analyses of ROS production. The slides were imaged by Laser Scanning Confocal Microscope (FLUOVIEW FV3000, Olympus Co., Tokyo, Japan).

2.7 Isolation and extraction of total RNA

Isolate RNA from cell lysates employing Trizol RNA separation reagent (Sigma-Aldrich Chemical Co.) and extract total RNA. Add 1 ml of Trizol reagent to each well and react for five minutes. Centrifuge the lysates from each well at 14,000×g for fifteen minutes, and transfer the supernatant into another tube. For precipitating RNA, add equal volumes of isopropanol and mix them. And then centrifuge the mixture at 13,000×g for ethanol solution 75% (v/v) and centrifuge it at 12,000×g for five minutes. Ultimately, dry the RNA particles and dissolve them in a buffer solution treated employing 50 µL diethylpyrocarbonate (DEPC).

2.8 Detection of mRNA gene expression employing qRT-PCR

AceQTM qPCR SYBR Green Master Mix (High ROX Premixed; Agilent Corp., CA, USA) was employed for qRT-PCR analyses. The reaction solution was prepared for containing 10µL 2×SYBR qPCR mixture, 0.1 µL forward and reverse primers, 1 µL RNA template, 2µL of 50×ROX reference dye, and deionized distilled water for achieving a total volume of 20 µL/well. Establish qPCR-PCR machine (ABI StepOnePlus TM, Thermo Fisher Scientific, MA, USA). First, set the initial denaturation temperature to 95℃ for fifteen minutes for one cycle. Second, the denaturation at 95°C, annealing at 60°C and extension at 72°C lasted for ten, twenty and thirty seconds, separately. The whole PCR stage consists of forty cycles. The designed 5'- 3' forward and reverse primers were displayed in S-1.

2.9 Western blotting

Lyse Cells employing RIPA lysis buffer containing phosphatase inhibitor mixture and protease inhibitor mixture. Collect lysed cells and centrifuge them at 1500xg for 15min. Transfer the supernatants to 1.5ml e-tube as entire cell lysate. The protein con-centration in the entire-cell lysate was determined by the BCA approach. The same amount of protein was transferred to the NC membrane after SDS- polyacrylamide gel electrophoresis. Block the NC membranes employing 5% skim milk or 1% BSA in TBST. After diluted with 1% BSA solution or 5% skimmed milk, conduct the antibody reaction overnight at 4℃, and then rinse the NC membrane employing TBST three times for five minutes each time. Secondly, couple the secondary antibody with HRP at room temperature and detected on the NC membrane for 1 hour. After the membrane was rinsed employing TBST, the chemiluminescence substrate reaction of HRP was conducted. The Biological Imaging System iBright CL750 (Thermo Fisher Scientific, MA, USA) imaged and photographed the protein band of each target. Protein bands were densitometric analyzed using Thermo Fisher ABI qubit4 (Thermo Fisher Scientific, MA, USA).

Homogenize the cells in precooled RIPA buffer (newly added protease inhibitor) and centrifuge it at 1500×g for 15min at 4℃. The collected supernatant was whole cell lysate. Prepare cytoplasmic and nucleocytoplasmic proteins employing protein extraction kits on basis of the manufacturer's instructions. Employ the BCA kit for determining the protein content. Apply the protein to 10% SDS-PAGE and electrophoretically separate it before transferring it to NC membrane (Pull company, USA). Seal the membrane in 5% skimmed milk powder for 60 minutes. Then the membrane was mixed with primary antibody (MMP-1 (E9S9N) Rabbit mAb, MMP-9 (D6O3H) XP® Rabbit mAb, MMP-2 (D8N9Y) Rabbit mAb, SAPK/JNK Antibody, Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb, COX-2 (D5H5) XP® Rabbit mAb, P38 MAPK (Thr180/Tyr182) (12F8) Rabbit mAb, Phospho-P38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb, p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb, GAPDH (D16H11) XP® Rabbit mAb, NF-κB p65 (D14E12) XP® Rabbit mAb #8242,β-Actin (13E5) Rabbit mAb and horseradish peroxidase (HRP)-binding secondary antibody (goat anti-rabbit or goat anti-mouse), separately. Display the protein bands through enhanced chemiluminescence reagent and quantify them through AlphaEaseFC software. The GAPDH was employed as the internal standard.

2.10 Statistic analyses

Repeat every value at least 3 times and exhibit outcomes as average ± SD. ANOVA and Duncan multiple comparison tests were employed for statistical analyses, and SPSS 25.0 statistic analyses software was employed. Define p value below 0.05 as statistically significant.

3.Results

3.1 Effect of DSW on cell viability of HaCaT cells induced by UVB

In this study, how DSW affects cell viability of HaCaT keratinocytes induced by UVB was exhibited. As the Fig. 1 showed, it can be confirmed that DSW of 200m and 800m in 600,1200 and 1800 hardness ranges enhanced cell viability according to the slight repression of UVB-induced cell proliferation. On the contrary, DSW decelerated UVB-induced cell death in a hardness-dependent way, what can be seen from the cell viability of UVB-induced HaCaT. Moreover, this impact was related to the reduction of ROS expression in HaCaT cells induced by UVB. These outcomes imply DSW may improve cell viability by inhibiting the inflammatory response.

3.2 Effect of DSW on SOD, CAT, GSH-Px and MDA levels in UVB-induced HaCaT cells

UVB-induced ROS generation holds the key to skin photoaging. The production of ROS was detected by immunofluorescence, and the level of ROS was expressed by optical average. In the immunofluorescence image of skin tissue, red indicated positive expression and blue indicated the nucleus.

Excessive ROS generation is related ultraviolet irradiation, which caused OS and inflammatory response. Antioxidant enzymes like GSH-Px, CAT, and SOD remove excess ROS and restore cellular state from immune stress. Compared with normal control group, the GSH-Px, CAT and SOD antioxidant enzymes in model group dropped by 30%, 50% and 11%, respectively (Table 1). In comparison to the model group, DSW of various hardness and depth could obviously raise the drop of GSH-Px, CAT, and SOD antioxidant enzymes induced by UVB. However, no obvious difference existed in the three major antioxidant enzymes between the D800 group (HL, HM and HH) and the D200 (HL, HM and HH).

Excessive ROS can lead to lipid peroxidation, which leads to oxidative damage of cells. MDA is a by-product of lipid peroxidation, which is also induced by ROS. Table 1 exhibited MDA level in model group was obviously dropped by 30% in comparison to normal group. In comparison to model group, the six UVB-DSW treatment groups could significantly inhibit the increase of MDA induced by UVB (P < 0.05). Nevertheless, no statistically obvious differences were discovered between depth of 200m and 800m in same hardness group.

The outcomes exhibited that DSW effectively lessened the OS of HaCaT keratinocytes by inhibiting lipid peroxidation and antioxidant enzyme activity. Moreover, the activity of DSW was significantly correlated with its hardness, while the activity was not significantly correlated with its depth.

Table 1

Effect of DSW on the expression of oxidation-related enzymes in HaCaT cell line
Group	Hardness	SOD (U/mg prot)	CAT (U/mg prot)	GSH-Px (U/mg prot)	MDA (U/mg prot)
Normal	0	47.85 ± 2.98	2.23 ± 0.17	49.35 ± 3.27	3.42 ± 0.08
Model	0	32.13 ± 2.93^#	1.11 ± 0.15^#	42.15 ± 1.95^#	5.01 ± 0.13^#
HL D200	600	42.11 ± 0.59^*	1.39 ± 0.25^*	43.67 ± 1.83^*	4.46 ± 0.22^*
HL D800	600	43.26 ± 1.43^*,b	1.45 ± 0.16^*,b	44.50 ± 0.50^*,b	4.37 ± 0.25^*,b
HM D200	1200	44.10 ± 2.12^**	1.88 ± 0.18^**	48.54 ± 1.58^**	3.94 ± 0.12^**
HM D800	1200	45.95 ± 4.01^**,b	1.92 ± 0.15^**,b	49.05 ± 1.99^**,b	3.71 ± 0.04^**,b
HH D200 HH D800	1800 1800	41.12 ± 1.75^* 42.32 ± 1.75^*,b	1.32 ± 0.16^* 1.37 ± 0.14^*,b	42.25 ± 1.25^* 43.17 ± 1.35^*,b	4.41 ± 0.10^* 4.36 ± 0.15^*,b
Mg + Ca	1200	43.75 ± 1.45^**,b	1.78 ± 0.12^**,b	47.98 ± 0.72^**,b	4.08 ± 0.17^**,b
Exhibit data as average ± SD (n = 6). Significant differences exhibited in the data of different letters in the same column.^#P < 0.05, ^##P < 0.01 and ^###P < 0.005 only in comparison to normal control group, P < 0.05, P < 0.01 and **P < 0.005 only compared to model control group, ^b no sognificant, group D800 compared with group D200 of the same hardness.
What can be seen from Fig. 2, the ROS level in the model control group obviously exceeded that in the normal control group. Hardness 600, 1200, and 1800 groups had a significant repression on ROS production. Among them, the inhibition effect was most obvious in HM group of 1200 hardness. It shows that appropriate seawater hardness can limit ROS production and protect the skin from OS. There was a quantitative dependence between DSW activity and hardness.

3.3 Effect of DSW on MMPs levels in HaCaT cells induced by UVB

Photoaging skin was exhibited to contain high levels of matrix metalloproteinases, regarded to be a marker of photoaging resulting from its function of collagen digestion ^28–31. Since the results in Fig. 3 showed that DSW treatment improved UVB-induced collagen destruction, we were interested in assessing the photoprotective impact of DSW on the expression of matrix metalloproteinases formed in HaCaT cells lines after UVB exposure. The density analysis of Western blot bands showed that UVB exposure induced up-regulating MMP-2 and MMP-9 in comparison to cells not exposed to UVB (Fig. 3). It was found that the culture of DSW decreased the expression of MMP-9, MMP-2, and MMP-1 in the HaCaT cell line induced by UVB. Besides, previous studies exhibited inflammation was a key mediator of light load, and lipid mediator COX-2 was activated under ultraviolet irradiation ^{32, 33}. Here, we discovered DSW featured a similar repression on UVB-induced COX-2 expression. Therefore, DSW inhibits the expression of COX-2 or MMPs induced by UVB, further supporting the view that DSW features an anti-aging effect.

3.4 Effects of DSW on UVB induced TNF in HaCaT cells-α And IL-6 expression

Effect of DSW on the expression and distribution of proinflammatory cytokines in mouse skin induced by UVB immunohistochemical staining of skin showed IL-6 and TNF-α positive signals were brown. The results showed that in the model control group, the levels of IL-6 and TNF-α (Fig. 4) obviously exceeded those in the normal control group (P < 0.05). In comparison to the model control group, the levels of TNF-α and IL-6 dropped obviously in a dose-dependent method (P < 0.05). It’s suggested that DSW can lessen inflammatory reactions and prevent skin from photoaging.

3.5 Effect of DSW on MAPK signaling pathway in HaCaT cells induced by UVB

As shown in Fig. 5, UVB exposure raised the phosphorylation of p38 MAPK (p-p38) proteins, ERK (p-ERK), and JNK (p-JNK) in comparison to the normal control group. In comparison to the model control group, DSW could obviously curb the expression of p-p38 proteins, p-ERK and p-JNK induced by UVB (P < 0.05). No significant alteration existed in the expression of JNK, ERK, and p38MAPK in each treatment group.

Figure 5A. The effect of DSW on the expression of MAPKs in HaCaT keratinocytes irradiated by UVB was detected through Western blot. B. The protein expression level of MAPKs was analyzed quantitatively by Image Lab statistical software. After 24 hours of cell adhesion, the model, DSW with different hardness and 2% FBS were added to DMEM medium, and UVB irradiation was carried out at a distance of 15 cm; Then, further culture the cells for 24 hours. Data were exhibited as average ± SD (n = 6). Significant differences existed in the data of different letters in the same chart (*P < 0.05, **P < 0.01 and ***P < 0.005 in comparison to model control group only, ^##P < 0.01 MC in comparison to NC, ^aP<0.01 HM in comparison to Mg + Ca, ^bP no significant, HM compared with Mg + Ca)

3.6 Effect of DSW on NF-κB in UVB-induced HaCaT cells

NF-κB constitutes a downstream target of the MAPK signal pathway³⁴. Figure 6 exhibited that the nuclear level of NF-κB in the model control group obviously exceeded that of the normal control group, while DSW-1600 and DSW-2400 treatment groups obviously curbed the nuclear translocation of NF-κB expression by 34.51% and 55.26%, separately. The repression of DSW on UVB-induced translocation of NF-κB from the cytoplasm to the nucleus can prevent the invasion of skin inflammation.

3.1 Effect of DSW on cell viability of HaCaT cells induced by UVB

3.2 Effect of DSW on SOD, CAT, GSH-Px and MDA levels in UVB-induced HaCaT cells

Table 1

Effect of DSW on the expression of oxidation-related enzymes in HaCaT cell line
Group	Hardness	SOD (U/mg prot)	CAT (U/mg prot)	GSH-Px (U/mg prot)	MDA (U/mg prot)
Normal	0	47.85 ± 2.98	2.23 ± 0.17	49.35 ± 3.27	3.42 ± 0.08
Model	0	32.13 ± 2.93^#	1.11 ± 0.15^#	42.15 ± 1.95^#	5.01 ± 0.13^#
HL D200	600	42.11 ± 0.59^*	1.39 ± 0.25^*	43.67 ± 1.83^*	4.46 ± 0.22^*
HL D800	600	43.26 ± 1.43^*,b	1.45 ± 0.16^*,b	44.50 ± 0.50^*,b	4.37 ± 0.25^*,b
HM D200	1200	44.10 ± 2.12^**	1.88 ± 0.18^**	48.54 ± 1.58^**	3.94 ± 0.12^**
HM D800	1200	45.95 ± 4.01^**,b	1.92 ± 0.15^**,b	49.05 ± 1.99^**,b	3.71 ± 0.04^**,b
HH D200 HH D800	1800 1800	41.12 ± 1.75^* 42.32 ± 1.75^*,b	1.32 ± 0.16^* 1.37 ± 0.14^*,b	42.25 ± 1.25^* 43.17 ± 1.35^*,b	4.41 ± 0.10^* 4.36 ± 0.15^*,b
Mg + Ca	1200	43.75 ± 1.45^**,b	1.78 ± 0.12^**,b	47.98 ± 0.72^**,b	4.08 ± 0.17^**,b
Exhibit data as average ± SD (n = 6). Significant differences exhibited in the data of different letters in the same column.^#P < 0.05, ^##P < 0.01 and ^###P < 0.005 only in comparison to normal control group, P < 0.05, P < 0.01 and **P < 0.005 only compared to model control group, ^b no sognificant, group D800 compared with group D200 of the same hardness.

What can be seen from Fig. 2, the ROS level in the model control group obviously exceeded that in the normal control group. Hardness 600, 1200, and 1800 groups had a significant repression on ROS production. Among them, the inhibition effect was most obvious in HM group of 1200 hardness. It shows that appropriate seawater hardness can limit ROS production and protect the skin from OS. There was a quantitative dependence between DSW activity and hardness.

3.3 Effect of DSW on MMPs levels in HaCaT cells induced by UVB

3.4 Effects of DSW on UVB induced TNF in HaCaT cells-α And IL-6 expression

3.5 Effect of DSW on MAPK signaling pathway in HaCaT cells induced by UVB

3.6 Effect of DSW on NF-κB in UVB-induced HaCaT cells

As the largest organ of the human body, the skin is often affected by a variety of external pressures. Therefore, it is very important for the body to resist these harmful pressures. UV is the main source of skin damage. Continuous exposure to UVB can lead to DNA damage, gene mutation, and the development of skin cancer. Long-run exposure to UVB radiation causes skin photoaging with the formation of rough, dry, loose, and wrinkled skin. Previous studies³⁵ have shown that skin inflammation caused by long-term UVB irradiation is associated with the development of skin photoaging. The investigation was aimed at studying the preventive impact of DSW on UVB-induced photoaging by light damage HaCaT cell model and to explore its possible mechanism.

Oxidation pressure can be produced by internal resources and the external environment, resulting in some primary damage caused by peroxide. Lack of antioxidants will cause OS and excessive free radicals, peroxide membrane, and even DNA mutation, and affect the structural and functional abnormalities of proteins. ROS attack and react with stable skin cell molecules, resulting in cross-linking of elastin and collagen, while lessening the self-healing ability of the skin³⁶. In this study, as Fig. 1 and Fig. 2 showed, we researched the antioxidant effects and ability of reducing ROS production induced by UVB. The beneficial effects of DSW may include inhibiting the excessive production of ROS under UVB irradiation.

Generally speaking, the human body has three antioxidant enzymes: CAT, GSH-Px, and SOD. They are the first antioxidant defense system for removing excess free radicals and repairing oxidative damage. More and more evidence exhibited that the pathogenesis of UVB light injury was associated with OS and redundant ROS production, seriously destroying the skin's antioxidant defense system. Lipid peroxidation constitutes a key source of OS, which is obviously raised in UVB-induced HaCaT cells. Malondialdehyde (MDA) serves as a lipid peroxidation product caused by UVB. It’s broadly considered a key biological marker of OS. In this study, as shown in Table 1, DSW can significantly improve the decrease of GSH-Px, CAT, SOD antioxidant enzyme activities and MDA level increase caused by UVB.

The main reason of skin light injury is the degradation and destruction of collagen, the main structural component of the dermal extracellular matrix. Overexpression of MMPs can degrade collagen, causing skin wrinkles and sagging. In addition, because UVB irradiation inhibits skin hydration, the skin surface will become dull and rough, and the skin epidermis is irregularly thickened, which is dubbed epidermal hyperplasia, causing skin wrinkles and roughness³⁷. In the investigation, DSW obviously curbed the protein expression of MMPs (Fig. 3). It showed DSW had a protective effect on photoaging caused by UVB.

As key regulators of extracellular signals from the cell surface to nucleus, mitogen-activated protein kinases (MAPKs), comprising p38, ERK, and JNK, hold the key to cell differentiation, proliferation, and apoptosis. UVB-induced OS holds the key to activating the MAPK path. While JNK and p38 are activated by cytokine receptors and different environmental stimuli (like ultraviolet), ERK is mainly activated by growth factor receptors. Previous studies exhibited that UVB induced OS mediates the phosphorylation level of MAPKs, encompassing JNK, ERK, and p38, related to photoaging³⁸. Nevertheless, no investigations have identified DSW plays an anti-aging role by inhibiting the MAPK signal pathway. Therefore, this study suggests that DSW may inhibit the activation of the UVB-induced MAPK signaling path by inhibiting UVB-induced OS, a key target for preventing and treating photoaging. It’s exhibited that phosphorylation of MAPK protein mediates the downstream event NF-κB. ERK and p38MAPK proteins were shown to regulate NF-κB activation. NF-κB is a transcription factor, holding the key to all tumor and carcinogenesis stages. It can be activated by UVB and other drugs to produce ROS. MAPK and NF-κB signaling paths regulate MMPs expression. Previous investigations have reported the activation of NF-κB and MAPK constitutes the main reason to stimulate and accelerate the expression of MMPs. In this study, DSW treatment attenuated UVB-induced MMPs expression by inhibiting MAPK protein phosphorylation and NF-κB translocation to the nucleus. In this study, we found that DSW reduced the phosphorylation of NF-κB and MAPKs in UVB exposed HaCaT keratinocytes (Fig. 5). In addition, cytoplasmic NF-κB expression was also downregulated (Fig. 6). Therefore, the results showed that DSW decreased the activity of HaCaT keratinocytes irradiated by UVB by inducing the expression of MMPs through NF-κB and MAPKs signaling pathway, so as to activate autophagy. Activation of NF-κB up-regulates the expression of inflammatory cytokines, encompassing IL-6 and TNF-α. IL-6 and TNF-α are considered to be key inflammatory intensity regulators. In this study, as shown in Fig. 4, DSW treatment effectively curbed the mRNA expression of IL-6 and TNF-α and inhibited NF-κB activation and nuclear translocation in HaCaT keratinocytes induced by UVB.

Here, we investigated the effects of 200m and 800m DSW depth of 600,1200,1800 hardness, and Mg + Ca in HaCaT keratinocytes irradiated by UVB. The results showed that both DSW and Mg + Ca alleviate UVB-induced cell death. However, no obvious differences existed in seawater activity at different depths of 200 and 800m. DSW is rich in Mg. Ca constitutes another main component in DSW. The ratio of calcium and magnesium ions is 3:1. For studying the anti-inflammatory and anti-photoaging impact of Mg, Ca and other trace elements in DSW, group Mg + Ca with the same hardness contained the same proportion (3:1) of magnesium and calcium as the reference. As the Fig. 1 showed, the DSW significantly improved the drop of cell survival induced by UVB, and 1200 hardness DSW at 200m and 800m significantly improved the cell survival rate reduction, which was significantly better than Mg + Ca group. Nevertheless, no significant differences exist between the activity of seawater at a depth of 200m and 800m, this may due to the unique properties of deep water, such as cleanliness, do not play a dominant role in its display of anti-photoaging activity. As Fig. 2 and Table 1 showed, both the group of DSW HM and Mg + Ca could obviously improve the expression of oxidation-related enzymes and downregulation the expression of ROS. As Fig. 3 showed, DSW and Mg + Ca all can up-regulation/down-regulation the expression of MMPs and COX-2. DSW had significantly better regulatory effects on expression of COX-2, MMP-9, MMP-1 protein than Mg and Ca solutions. As shown in Fig. 4 and Fig. 5A, Mg + Ca also down-regulation the MAPKs expression and nucleus-NF-κB in HaCaT keratinocytes radiated by UVB. Therefore, the result suggests the major role of Mg and Ca on suppression inflammatory response of HaCaT keratinocytes irradiated by UVB. However, under the same concentration of magnesium and calcium ions, the anti-inflammatory and anti-photoaging activity of DSW is significantly better than Mg + Ca solution. This may be due to the DSW rich in a variety of trace element ions.

Collectively, as Fig. 7 showed, NF-κB and MAPK signaling paths may be potential targets to mediate MMPs expression in HaCaT keratinocytes irradiated by UVB and hold the key to the protective impact of DSW on the light injury. The conclusion shows that DSW may be used in skin health care by reducing skin cell damage caused by ultraviolet radiation.

The investigation proved the DSW from South China Sea could ameliorate the inflammation, collagen degradation and aging induced by UVB. It may be mediated by regulating MAPK and NF-κB signaling paths, inhibiting UVB-induced OS and inflammatory response, and MMP-1 expression. Therefore, DSW may be a potential skin health anti-aging agent.

Conflict of interest

The corresponding author states on behalf of all authors no conflict of interest exists.

Acknowledgement

The project has been supported by the Central Research Institutes Project (K-JBYWF-2018-T02, K-JBYWF-2019-T06), Tianjin Science and Technology Plan Project (20JCYBJC00830) and the Open Project of State Key Laboratory of Food Nutrition and Safety of the Ministry of Science and Technology of Tianjin University (No. SKLFNS-KF-202007).

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No competing interests reported.

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The protects of deep sea water against photoaging of HaCaT keratinocyte induced by UVB via suppression MMPs and MAPKs/NF-κB signaling pathway

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Abstract

Figures

1. Introduction

2. Materials And Method

2.1 Chemicals and reagents

2.2 Fabrication of deep sea water

2.3 Cell treatment and UVB irradiation

2.4 Cell viability assay

2.5 SOD, CAT, GSH-Px, and MDA determination

2.6 Immunofluorescence analyses

2.7 Isolation and extraction of total RNA

2.8 Detection of mRNA gene expression employing qRT-PCR

2.9 Western blotting

2.10 Statistic analyses

3.1 Effect of DSW on cell viability of HaCaT cells induced by UVB

3.2 Effect of DSW on SOD, CAT, GSH-Px and MDA levels in UVB-induced HaCaT cells

3.3 Effect of DSW on MMPs levels in HaCaT cells induced by UVB

3.4 Effects of DSW on UVB induced TNF in HaCaT cells-α And IL-6 expression

3.5 Effect of DSW on MAPK signaling pathway in HaCaT cells induced by UVB

3.6 Effect of DSW on NF-κB in UVB-induced HaCaT cells

3. Results

3.1 Effect of DSW on cell viability of HaCaT cells induced by UVB

3.2 Effect of DSW on SOD, CAT, GSH-Px and MDA levels in UVB-induced HaCaT cells

3.3 Effect of DSW on MMPs levels in HaCaT cells induced by UVB

3.4 Effects of DSW on UVB induced TNF in HaCaT cells-α And IL-6 expression

3.5 Effect of DSW on MAPK signaling pathway in HaCaT cells induced by UVB

3.6 Effect of DSW on NF-κB in UVB-induced HaCaT cells

4. Discussion

Conclusion

Declarations

References

Additional Declarations

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