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


 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.


Introduction
Skin aging is a multi-system degradation course involving skin and skin support systems 1 . Exposure to ultraviolet light may lead to oxidative stress (OS), immunosuppression, in ammation, 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][3][4] . In humans, photoaging is characterized by ne and rough wrinkles, dry and rough skin, sun skin, sallow color, poor elasticity recoil, and impaired wound healing [5][6][7] . The current study suggests that the accumulation of ROS reduces the body's salubrity as oxidative damage is a factor leading to aging 8 .
However, long-term exposure to ultraviolet radiation can cause severe adverse impacts on skin structure and function, eventually leading to photoaging and even skin cancer 9 .
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 . In ammation and its induced in ammation hold the key to the process of human skin photoaging. However, ROS levels are signi cantly increased under environmental stresses, such as UVB or heat exposure, resulting in signi cant impairment of cell function and structure. Previous studies 11 have exhibited that UV-induced ROS activates lipid mediators, comprising cyclooxygenase-2 (COX-2). In addition, exposure of ultraviolet light UV increases the proin ammatory mediators released by diverse skin cells, leading to the activation of matrix metalloproteinases (MMPs) and matrix-degrading enzymes 12 . However, in ammation-activated various matrix degradation matrix proteinases lead to accumulation of non-functional matrix components and abnormal matrix degradation, causing photoaging 13 . Therefore, previous studies 11,14 have exhibited MMPs can be employed as the main markers of wrinkles and skin in ammation caused by UVB.
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 DSW 25 . Nevertheless, to our knowledge, how DSW affects UVB-induced skin injury hasn't been completely clari ed 26 . Hence, we evaluated how DSW affects UVB-induced HaCaT keratinocyte injury and further explored the molecular biological regulation mechanism of DSW on UVB-induced in ammation and photoaging of HaCaT cells.

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 ltration 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 MgSO 4 and CaCl 2 (Mg:Ca = 3:1) was fabricated as Mg + Ca group (hardness 1200) as well.

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 strati ed employing PBS. PL-S 5W/2P UVB lamp (wavelength 311nm, Philips, Netherlands) was employed for UVB irradiation. The irradiation distance was 10cm (16.7mJ/cm 2 ) and irradiated in the cassette. Cells were cultured in DMEM without FBS for 4h. The UVB intensity at 10cm distance was 0.1mW/cm 2 .

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 nal volume was 200 µL. Subsequently, alter the medium to 200µL 0.5 mg mL − 1 MTT solution and incubate them in 5% (v/v) CO 2 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 Scienti c 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).

SOD, CAT, GSH-Px, and MDA determination
Plate 5×10 4 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.

Immuno uorescence analyses
Immuno uorescence 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 1×10 5 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, immuno uorescence 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).

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 ve minutes. Centrifuge the lysates from each well at 14,000×g for fteen 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 ve 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 Scienti c, MA, USA). First, set the initial denaturation temperature to 95℃ for fteen 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.

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 ve 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 Scienti c, MA, USA) imaged and photographed the protein band of each target. Protein bands were densitometric analyzed using Thermo Fisher ABI qubit4 (Thermo Fisher Scienti c, MA, USA).

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. De ne p value below 0.05 as statistically signi cant.

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 con rmed 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 in ammatory response.

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 immuno uorescence, and the level of ROS was expressed by optical average. In the immuno uorescence image of skin tissue, red indicated positive expression and blue indicated the nucleus.
Excessive ROS generation is related ultraviolet irradiation, which caused OS and in ammatory 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 byproduct 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 signi cantly 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 signi cantly correlated with its hardness, while the activity was not signi cantly correlated with its depth. Exhibit data as average ± SD (n = 6). Signi cant 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 sogni cant, 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 signi cant 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.

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][29][30][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 in ammation 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.

Effects of DSW on UVB induced TNF in HaCaT cells-α And IL-6 expression
Effect of DSW on the expression and distribution of proin ammatory 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 in ammatory reactions and prevent skin from photoaging.

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 con rmed that DSW of 200m and 800m in 600,1200 and 1800 hardness ranges Excessive ROS generation is related ultraviolet irradiation, which caused OS and in ammatory 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 byproduct 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 signi cantly 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 signi cantly correlated with its hardness, while the activity was not signi cantly correlated with its depth. Exhibit data as average ± SD (n = 6). Signi cant 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 sogni cant, 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 signi cant 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.

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][29][30][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 in ammation 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.

Effects of DSW on UVB induced TNF in HaCaT cells-α And IL-6 expression
Effect of DSW on the expression and distribution of proin ammatory 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 in ammatory reactions and prevent skin from photoaging.

Effect of DSW on MAPK signaling pathway in HaCaT cells induced by UVB
As shown in Fig

Effect of DSW on NF-κB in UVB-induced HaCaT cells
NF-κB constitutes a downstream target of the MAPK signal pathway 34 . 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 in ammation.

Discussion
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 studies 35 have shown that skin in ammation 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 skin 36 . 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 bene cial 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 rst 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 signi cantly 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 roughness 37 . 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 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 in ammatory cytokines, encompassing IL-6 and TNF-α. IL-6 and TNF-α are considered to be key in ammatory 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 signi cantly improved the cell survival rate reduction, which was signi cantly better than Mg + Ca group. Nevertheless, no signi cant 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 signi cantly 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 downregulation 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 in ammatory response of HaCaT keratinocytes irradiated by UVB. However, under the same concentration of magnesium and calcium ions, the anti-in ammatory and anti-photoaging activity of DSW is signi cantly 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.

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

Declarations Con ict of interest
The corresponding author states on behalf of all authors no con ict of interest exists. Figure 1 Effect of DSW on the activity of UVB-induced HaCaT keratinocyte. (A) Cells were exposed to UVB 15cm away and DMEM conditioned medium supplemented with different hardness magnesium calcium solutions, and different depth and hardness of DSW and 2% FBS; then, further culture the cells for 24h. Exhibit data as the average ± SD of three independent experiments. #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 with model control group, b no sogni cant, group D800 and Mg+Ca compared with group D200 of the same hardness HM. D200 means Depth of 200m, D800 means depth of 800m, HL means hardness of 600, HM means hardness of 1200, HH means hardness of 1800.

Figure 2
Effect of DSW on ROS expression in HaCaT keratinocytes irradiated by UVB. The cells were placed in a 60 mm dish. After the cells adhered to the wall for 24 hours and were irradiated with UVB at a distance of 15 A. Effects of DSW on the expression of MMPs and COX-2 in HaCaT keratinocytes irradiated by UVB were detected by Western blot. B. The protein expression levels of MMP-1 (a), MMP-2 (b), MMP-9 (c), and COX-2 (d) were 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. Exhibit data as average ± SD (n=6). Signi cant differences existed in the data of different letters in the same chart (*P < 0.05, **P < 0.01 and ***P < 0.005 only in comparison to model control group, ##P<0.01 MC in comparison to NC, aP<0.01 HM in comparison to Mg+Ca, bP no signi cant, HM compared with Mg+Ca) Figure 4 Effect of DSW on IL-6 and TNF-α mRNA expression in UVB-radiated HaCaT keratinocyte was examined by Q-PCR. 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). Signi cant 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 signi cant, HM compared with Mg+Ca) 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). Signi cant 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 signi cant, HM compared with Mg+Ca) Figure 6 A. Effects of DSW on cytosol-NF-κB p65 and nucleus-NF-κB p65 expressions in UVB-radiated HaCaT keratinocyte was examined through western blot analyses. 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). Signi cant 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 signi cant, HM compared with Mg+Ca)