Protective Activity of Alpha-Mangostin against UVB-Induced Injury in HaCaT Cells by Modulating the Ceramide and MAPK and NF-κ B Signaling Pathways

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Introduction
Tere are various factors that afect the aging of the skin, such as ultraviolet (UV), air pollution, oxidative stress, and heredity, among which photoaging plays a principal role in skin aging, and skin photoaging approximately accounts for 80% in skin aging [1].A major reason of skin photoaging is exposure to UV in sunlight, which alters the composition of skin cells and causes the loss of components of the extracellular matrix (ECM), thereby damaging the skin [2,3].Te UV can be partitioned into three regions depending on diferent wavelengths, respectively, including ultraviolet A (UVA) (320-400 nm), ultraviolet B (UVB) (290-320 nm), and ultraviolet C (UVC) (200-290 nm).Since the great majority of UVC is absorbed by the ozone layer before reaching the earth, it can be concluded that the two critical types of UV that cause skin damage are UVA and UVB.In particular, UVB which is capable of causing skin sunburn has about 1000 times genotoxicity than UVA [4].Constant exposure to UVB over a long period results in damages such as skin erythema, sunburn, oxidative damage, apoptosis, infammation, and sometimes even skin cancer [2,3,5].
Alpha-mangostin (α-MG) is a major xanthone compound widely found in mangosteen pericarp [13].It was confrmed to possess various bioactive functions including anti-infammatory, antiapoptosis, anticancer, and antioxidant efects [14][15][16], and it has also been identifed as an inhibitor of acid sphingomyelinase (ASM) [17].ASM, which hydrolyzes sphingomyelin (SM) to Cers and phosphorycholine, is considered to be a key target of Cer production upon UV irradiation [18,19].α-MG improves endothelial dysfunction in diabetic mice through the inhibition of Cer [20].Cers are important structural components of the epidermis, which plays a key role in maintaining homeostasis of the human body [20].In addition, Cers also act as an active second messenger, regulate keratinocyte proliferation and diferentiation, and enhance proinfammatory cytokines production [21].Although study has shown that α-MG has an antiphotoaging efect [22], mechanisms of α-MG in protecting the skin from UVB damage still lack in-depth study.Terefore, we committed to studying the protective efect of α-MG for HaCaT cells against UVB irradiation and its potential molecular mechanism.Tis study aimed to provide both a theoretical and experimental basis for the development and application of α-MG; thereupon, a ceramide that is a prominent manifestation in the process of photoaging is disclosed.
Chloroform was of HPLC grade and was obtained from Honeywell (AH049-4, Morris Tow., USA).Methanol was of HPLC grade and was purchased from Fisher chemical (A452-4, Shanghai, China).Ammonium hydroxide solution was provided by Sigma-Aldrich (05002-1L, Missouri, USA).Te UVB source employed is a Sigma SH4B light therapy device (Shanghai SIGMA High-tech Co., Ltd., Shanghai, China), with wavelength ranging between 311 and 313 nm and having a peak wavelength of 313 nm.

UVB Irradiation and Administration.
Dissolve the appropriate amount of α-MG in DMSO to prepare a 10 mM stock solution and then directly dilute the stock solution to the appropriate concentration with DMEM (complete medium).HaCaT cells were allowed to grow an appropriate abundance and then the treatment group was treated with α-MG at a concentration of 2 μM.After 2 h, the complete culture medium of DMEM (complete medium) was substituted with PBS and then the cells were irradiated in UVB (50 mJ/cm 2 ).Te PBS was removed after the UVB irradiation, and the culture of cells was sustained for 24 h in the complete medium for subsequent experiments.Te control group was treated with the same conditions but without any exposure to UVB irradiation or α-MG treatment, whereas the UV group was irradiated with UVB but did not receive α-MG treatment.
2.4.Cell Viability.After treatment, cells were incubated for 4 h in the culture medium with 10 μL of MTT (5 mg/mL).After removing the supernatant, 100 μL of DMSO was added into the complete DMEM.Finally, a plate reader (ST-360, Shanghai Kehua Biological Engineering Co., Ltd., Shanghai, China) was used to measure its absorbance at 490 nm.
2.5.Apoptosis.After treatment, cells and supernatant were collected to assay the apoptosis rates.Te annexinV-FITC apoptosis assay kit was used to perform the experiments following the manufacturer's instructions.Apoptotic and necrotic cells were measured using the EXFLOW fow cytometer (EXFLOW-206, Changzhou Bidake Biotechnology Co., Ltd., Jiangsu, China).

Real-Time Quantitative PCR (RT-qPCR).
After treatment, cells were collected and total RNA was extracted using TRIzol.After quality checking and purifcation, 2 μg of RNA was reverse transcribed to cDNA by using the reverse transcription kit.RT-qPCR was performed using SYBR Green Master Mix in a Bio-Rad IQ5 detection system.GAPDH is indexed as an internal reference, and at the same 2 Journal of Food Biochemistry time, three wells were set for each sample.Te 2 −△△Ct method was used to calculate the relative content of mRNA.Te upstream and downstream primer sequences of the gene are listed in Table 1.
2.7.Measurement of IL-6.After treatment, supernatants of HaCaT cells were collected and centrifuged at 3,000 rpm for 10 min.Te ELISA kit was used to measure IL-6 concentrations following the manufacturer's instructions.Te absorbance (A450) was measured using a plate reader with a detection sensitivity of at least 1.0 pg/mL.Te contents of IL-6 were calculated by interpolating the results onto a standard curve.
2.8.Immunoblotting.After treatment, cells were washed three times with PBS and collected in RIPA bufer for 30 min.

Statistical Analysis.
All the data were shown as the mean ± standard deviation (SD) and processed using IBM SPSS Statistics 19 software.Te two-independent-sample test was performed to assess diferences between the two groups.A level of P < 0.05 indicated statistically signifcant.

Efect of α-MG and UVB on Cell
Viability.Te efect of diferent concentrations of α-MG (2, 4, 6, 8, 10, 15, and 20 μM) on the viability of HaCaT cells was assessed by the MTT method.Te results showed that the IC 50 value of α-MG was 19.17 μM on HaCaT cells, and there was no cytotoxicity when its concentration was inferior or equal to 10 μM (Figure 1(a)).Te cell viability of cells irradiated with diferent UVB doses was assessed using the MTT assay.Like reported [25], we decided to create a cell model of photoaging under a median lethal irradiation intensity of 50 mJ/ cm 2 (Figure 1(b)).
To determine the potential protective efect that α-MG exerts on HaCaT cells, we performed MTT assay on HaCaT cells after exposure to UVB irradiation.After UVB exposure, the cell viability of HaCaT cells decreased strikingly.α-MG played a positive role in the proliferation of HaCaT cells.Compared with the UV group, α-MG at 2 μM enhanced viability of HaCaT cells by 10.24% (Figure 1(c)).
Tese observations indicate that α-MG has protective efects in UVB-induced HaCaT cells.

α-MG Inhibits the Expression of MMPs.
MMPs play a considerable role in the physiological mechanism of skin photoaging.Te action of UV is to transform the connective tissue of the skin by upregulating the expressions of MMPs which degrade collagen and other ECM proteins [26,27].MMP-1 is a member of the MMPs in the collagenase family.After being activated, MMP-1 further degrades the collagens of type I and type III that has been degraded beforehand by MMP-2 and MMP-9, thereby provoking the degradation of collagen [26,28].Simultaneously, the gelatinolytic activity of MMP-9 is the central factor for skin damage and wrinkle formation due to UV [25,29].Terefore, the assessments on the expressions of MMPs have become preliminary in the process of screening agents against photoaging.In this study, we examined the mRNA levels of MMP-1, MMP-2, and MMP-9 through RT-qPCR.UVB irradiation dramatically increased the expressions of MMPs and α-MG treatment reversed this trend (Figures 1(d)-1(f )).Tis result indicates that α-MG prevents the loss of ECM and has a potential antiphotoaging efect.Tus, α-MG has the opportunity to be a candidate drug for the prevention and treatment of photoaging.

α-MG Inhibits Apoptosis.
Apoptosis is a key factor of cell growth and homeostasis that maintain normal cell proliferation [30].In this study, we explored the antiapoptotic properties of α-MG (Figure 2).As shown in Figures 2(a) and 2(b), UVB irradiation increased the percentage of apoptotic cells from 6.27% to 65.60%, which was remarkably reduced to 45.34% by α-MG.Te results showed that pretreatment of HaCaT cells with α-MG was capable of attenuating apoptosis induced in UVB efcaciously.
Te activation of caspase-9 is the major initial step of the caspase-dependent endogenous apoptotic pathway [31].Subsequently, downstream caspases or other caspases, including caspase-3, -6, and -7, are activated, eventually leading to cell death.Te caspase substrate proteins are degraded in this process, which is the evidence of the induction of caspase-dependent apoptosis [31,32].Te activation of caspase is manipulated by a variety of proteins, including members from the Bcl-2 family, such as Bcl-2 and Bax [32].Terefore, the balance between the proapoptotic Bax and the antiapoptotic Bcl-2 plays a determinant role in activating cascade reaction through inducing or inhibiting caspase, thereby cascading to initiate the intrinsic apoptotic pathway.For the purpose of further exploring the inhibiting mechanism of α-MG inhibiting cell apoptosis, we used RT-qPCR and western blot to examine the mRNA and protein expression levels of Bcl-2, Bax, and caspase-3.As shown in Figures 2(c)-2(g), compared with the control group (C), the ratio of Bax/Bcl-2 and the expression of caspase-3 in the UV group (U) were considerably increased.Compared with the U group, the ratios in the treatment group (T) were evidently decreased.Te variations between mRNA and protein levels are consistent.Te abovementioned results indicated that α-MG has an ability to resist UVB-induced apoptosis.

α-MG Decreases IL-6 and TNF-α Production.
Accumulating evidences indicate that after exposure to UVB, infammatory response causes skin damage due to its stimulation on the secretion of infammatory cytokines, including IL-6, TNF-α, and COX-2 [26,33].Similarly, in the present study, the HaCaT cells, one of the major targets of UVB, exhibited higher levels of proinfammatory cytokines when exposed to UVB.In addition, no matter what circumstances in which skin damage is caused, keratinocytes will be induced into excessive multiplication, which is closely related to the secretion of proinfammatory cytokines [34].Terefore, it is of great signifcance to investigate the anti-

α-MG Inhibits NF-κB Activation and MAPK
Phosphorylation.MAPK and NF-κB play pivotal roles in cellular responses to extracellular signals.Upon stimulation with predominantly pathological agents, MAPK modulates the NF-κB activity by modulating its transactivation.Te activated NF-κB complexes translocate from the cytoplasm to the nucleus and bind to the NF-κB target gene.Many studies have shown that UVB irradiation induces the activation of MAPK which can function as the upstream mediator of MMP-9 [35,36].It has also been reported that NF-κB increases MMP-9 expression in fbroblasts [37,38].Terefore, the phenomena of skin aging, such as UVBinduced MMP-9 expression, may be associated with the levels of phosphorylated MAPK and the activation of NF-κB.
It is well known that the NF-κB signaling pathway is also a classic infammatory pathway, and it plays a major role in the production of proinfammatory cytokines in response to UVB irradiation.Our research showed that the increase in NF-κB phosphorylation induced by UVB irradiation was signifcantly decreased by α-MG (Figures 3(d)-3(f )).Tese suggest that α-MG exerts photoprotection through alleviating the infammatory response to UVB irradiation by regulating NF-κB signaling.Te MAPK pathway-related proteins play a major role in the apoptosis and infammation in response to UV irradiation [8,9].Te increase in JNK, P38 MAPK, and ERK1/2 phosphorylation induced by UVB irradiation was signifcantly decreased by α-MG, and UVB and α-MG had no efect on nonphosphorylated JNK, P38 MAPK, and ERK1/2 (Figure 4).Taken together, these data demonstrate that α-MG protects HaCaT cells from UVB-induced apoptosis and infammation by regulating MAPK signaling (Figures 2-4).Tis suggested the possibility that α-MG might attenuate photoaging-associated changes in vivo and suppress the infammation of skin tissues.

α-MG Inhibits Cer Production.
Lipids are important structural and functional components of the skin.Alterations in the lipid composition of the epidermis are associated with infammation and apoptosis and can afect the barrier function of the skin [39,40].In this study, we investigated the changes in the lipid metabolism in HaCaT cells.By UPLC, we analyzed the molecular species of SM, Cer, glucosylceramide (GlcCer), and lactosylceramide (LacCer) in HaCaT cells in diferent groups.Te numbers of lipid identifcations are displayed in Figure 5(a).Te lipid heat map analysis in HaCaT cells showed the following (Figure 5(b)): UVB irradiation leaded to an increase in the level of SM and its metabolites, such as Cer, GlcCer, and LacCer.After administration, the SM and Cer contents decreased and the content of GlcCer increased, while LacCer had no signifcant change.Te research of Magnoni et al. showed that UVB irradiation increased the production of Cer in HaCaT cells, and there was a temporal correlation between the production of Cer and the apoptosis UVBinduced [41].It was also detected that the activation of neutral sphingomyelinase and ASM was activated when HaCaT cells were exposed to UVB [41].Moreover, Okudaira found that α-MG inhibited ASM with an IC50 value of 14.1 μM (5.8 μg/mL) [17].In this study, we observed that the content of Cer in the HaCaT cells UVB-induced increased.Te administration of α-MG reversed this phenomenon.Combined with the changes of SM, GlcCer, and LacCer preor postadministration, it can be known that the degradation of SM was alleviated reducing the formation of Cer because α-MG inhibited the activity of ASM.In the meantime, the content of GlcCer (Cer derivative) still increased after administration.Tis indicates that under the intervention of α-MG, Cer tends to be converted into LacCer, thereby reducing its content.Tese results showed that after administration, α-MG on the one hand reduced the generation of Cer and on the other hand accelerated the conversion of Cer and ultimately reduced the level of Cer in HaCaT cells, protecting cells from damage caused by UVB irradiation.
Subsequently, we further analyzed the results and plotted a Venn diagram (Figure 5(c)) (U group vs. C group and T group vs. U group, P < 0.05).Notably, Cer d18 : 1/24 : 1 (a type of ceramide) had a biological signifcance in pairwise comparisons.Te heat map of Cer (Figure 5(d)) and the relative content of Cer d18 : 1/24 : 1 (Figure 5(e)) once again confrmed the trend of Cer that UVB irradiation increased the content of Cer, and α-MG decreased the content of Cer.Cer, a lipid messenger, is one of the main mediators that regulate multiple pathways related to skin cell apoptosis and infammation [18,19].UVB irradiated promoted cell apoptosis and aggravated the degree of infammation, while the administration of α-MG reversed this phenomenon.It indicated that α-MG might inhibit cell apoptosis and infammation in photoaging by reducing the content of Cer.It has also been reported that Cer is the upstream mediator of  6 Journal of Food Biochemistry MAPK [10][11][12].Terefore, the activation of MAPK and NF-κB is related to the increase of Cer.Currently, research studies on Cer d18 : 1/24 : 1 basically focus on cardiovascular disease, diabetes, liver and kidney disease, and Alzheimer's disease [42][43][44].In human keratinocytes, we disclosed its changes and efects for the frst time.Tis provides a new target for other researchers to study skin photoaging.
However, this study lacks data to prove that it is a biomarker of skin photoaging.
In conclusion, the present study demonstrated the protective efects of α-MG on UVB-induced photoaging in HaCaT cells.α-MG has the potential to protect the skin   from UVB-induced photoaging, such as MMP-9 production, apoptosis-related proteins increase, and infammatory factors production, possibly through the inactivation of ceramide-MAPK-NF-κB signaling pathways (Figure 6).Te present study suggests that α-MG has a potential as therapeutic agents for the prevention and treatment of skin photoaging.However, because of the complex regulation of signaling pathways in cells, specifc intermediates and intermolecular interactions need to be in-depth studied.

Figure 5 :
Figure 5: α-MG reduced the levels of Cer.(a) Te metabolite statistics chart showed the 4 lipids of SM, Cer, GlcCer, and LacCer and their respective numbers.(b) After cluster analysis, the overall heat map of the four lipids in lipid metabolism was obtained.(c) Te Venn diagram was obtained after further analysis.A represented U group vs. C group; B represented T group vs. U group; P < 0.05. 1 represented Cer d18 : 1/24 : 1.(d) After cluster analysis, the heat map of Cer was obtained.(e) Te relative content of Cer d18 : 1/24 : 1 in each group was presented ( * * * P < 0.001 vs. the control group; # P < 0.05 vs. the UV group; n � 3; C: control group; U: UV group; T: treatment group).

Table 1 :
Sequence of primers used for RT-qPCR.