Sesamin attenuates UVA‐induced keratinocyte injury via inhibiting ASK‐1‐JNK/p38 MAPK pathways

Ultraviolet (UV) exposure‐stimulated reactive oxygen species (ROS) formation in keratinocytes is a crucial factor in skin aging. Phytochemicals have become widely popular for protecting the skin from UV‐induced cell injury. Sesamin (SSM) has been shown to play a role in extensive pharmacological activity and exhibit photoprotective effects.


| INTRODUC TI ON
The skin is responsible for protecting the body from external harmful factors, such as infections, chemical pollution, and different kinds of irradiation.Therefore, maintaining skin integrity is crucial for human health.Long-term ultraviolet (UV) irradiation is a key factor that induces skin injury and extrinsic skin aging.Despite being less energetic than UVB (290-320 nm), UVA (320-400 nm) radiation contributes significantly to photoaging by penetrating the deeper sections of the epidermis. 1It has been known that oxidative stress plays a major role in UV-induced skin injury and aging process.
Ultraviolet (UV) irradiation leads to the genesis of reactive oxygen species (ROS), which can disrupt the extracellular matrix (ECM), causing degradation of collagen and elastic fibers, as well as the collapse of the orderly network interwoven via long collagen fibrils, elastic fibers, glycoproteins, and glycosaminoglycans. 2,3Therefore, extrinsic aging is almost synonymous with "photoaging," which refers in particular to skin aging caused by UV irradiation and consequent ROS-triggered skin cell injury.Ultraviolet irradiation leads to the activation of mitogen-activated protein kinase (MAPK) p38, c-Jun amino-terminal kinase (JNK), and extracellular signal-regulated kinase, as well as the recruitment of c-Fos and c-Jun.[5] Antioxidants, as reducing agents, are commonly used to neutralize ROS to prevent the activation of the MAPK pathway and subsequent MMP production that degrades collagen, thereby alleviating skin aging. 62][13][14] Lignin, including SSM, contains UV-absorbing functional groups, and thus can be applied for UV protection. 15cent research indicates that SSM has antiphotodamage and antiinflammatory effects against UVB irradiation. 16However, the physiological outcomes of SSM against UVA-mediated skin oxidative injury and photoaging remain unclear.
In this study, we investigated the protective effects of SSM on UVA-irradiated human keratinocytes and evaluated its regulatory and ameliorative activities on cell injury, apoptosis, MMP expression, oxidative stress, and MAPK signaling pathway protein expression.

| UVA irradiation procedure
Freshly cultured HaCaT cells were exposed to UVA (320-400 nm) radiation at 8 J/cm 2 for 10 min when the cells reached 80%-90% confluence and then incubated with fresh culture medium for 24 h.

| Determination of cytotoxicity
The cytotoxicity of SSM in HaCaT cells was assessed using a CCK-8 assay kit (Beyotime, Shanghai, China).Briefly, 2 × 10 4 cells were seeded in 96-well plates and incubated with different concentrations of SSM for 24 h.Then, the CCK-8 reagent (10 μL for each well) was added and incubated at 37°C.The absorbance of each well was measured at 450 nm for 2 h to determine the proliferation ability and cytotoxicity of the cells in every well.The prosurvival effect of SSM on UVA-irradiated HaCaT cells was assessed using the CCK-8 assay kit (except for the UVA irradiation procedure), LDH assay kit, and morphological changes.For LDH release assays, briefly, after 24 h in culture according to UVA irradiation procedure, supernatants of HaCaT cells were collected after 24 h and treated with the LDH cytotoxicity assay kit (Beyotime, Shanghai, China) following the manufacturer's instructions.Finally, the absorbance was measured at 450 nm using a microplate reader.

| Determination of apoptosis
Apoptotic cells were stained with annexin V-FITC/propyridine iodide (PI), which was obtained using an annexin V-FITC apoptosis detection kit (Beyotime, Shanghai, China), and imaged by confocal microscopy.3 × 10 5 HaCaT cells per well were cultured in six-well plates for SSM pretreatment and UVA irradiation.After 24 h, the cells were collected, washed with PBS, and suspended in annexin V binding buffer.Then, they were fixed in 5 μL of annexin V-FITC and 5 μL of PI at 24°C for 15 min.Apoptosis in each well was detected using a laser scanning confocal microscope (Zeiss, Oberkochen, Germany).

| Determination of oxidative stress-related indicators
Specific reagent kits were used to identify the levels of oxidative stress-related indicators, including MDA, GSH, and SOD.A bicinchoninic acid assay kit (Beyotime, Shanghai, China) was used to quantify protein concentration.

| Determination of intracellular ROS
Intracellular ROS content was determined using an ROS assay kit (Beyotime, Shanghai, China) according to the manufacturer's instruction.Briefly, HaCaT cells were seeded on top of coverslips in 24-well plates and pretreated with SSM.After UVA irradiation, the cells were incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min and washed with PBS.Nuclei were stained with DAPI (Beyotime) for 20 min.The fluorescence in each well was measured using a laser scanning confocal microscope (Zeiss, Oberkochen, Germany).

| Western blotting
Western blotting was performed as previously described 17 to examine changes in protein content and/or phosphorylation.Briefly, after designated treatments, cells were lysed and centrifuged to obtain protein samples.These samples were loaded and separated on a 10%-12% SDS-PAGE gel and then transferred onto polyvinylidene fluoride membranes.The following antibodies were used for immunoblotting: anti-MMP-1, anti-MMP-9, anti-Bax, anti-Bcl-2, anti-ASK-1, anti-p-ASK-1, anti-JNK, anti-p-JNK, anti-p38, and anti-p-p38.

| Statistical analysis
Data are expressed as means ± SD of at least three independent experiments.Statistical differences between two groups were analyzed by Student's two-tailed unpaired t-test using the GraphPad Prism 8 software (GraphPad Software Inc.).A p-value <0.05 indicated a statistically significant difference.Statistical significance was set at # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the control group (C); and *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the UVA-irradiated cell group (M).

| SSM attenuated UVA-induced keratinocyte injury
To determine the cytotoxicity of SSM, the viability of HaCaT cells treated with gradient concentrations of SSM (0, 10, 20, 40, 80 and 160 μM) was detected using CCK-8 assay.Figure 2A shows that SMM exhibited negligible effects on HaCaT cells at 10, 20, 40, and 80 μM, and a mild toxicity at 160 μM.Sesamin also showed a proliferative effect at 40 μM.Therefore, we used 20 and 40 μM SSM as optimal doses for further experiments.To determine the protective effects of SSM on UVA-irradiated HaCaT cells, cell viability and LDH release were measured after UVA irradiation.UVA-irradiated cells showed a significant decrease in cell viability, whereas SSM treatment showed prosurvival effects on UVA-induced damage (Figure 2B).Microscopic images revealed that SSM reversed the UVA-induced variation in HaCaT cells (Figure 2C).Lactate dehydrogenase is a cytosolic enzyme released from damaged cells that can be used to evaluate the extent of cellular injury.We observed a statistically increased LDH release in UVA-irradiated cells, and a significant decrease in LDH release in 20 and 40 μM SSM-treated HaCaT cells (Figure 2D).Taken together, these results indicate that SSM pretreatment attenuates UVA irradiation-induced injury in HaCaT cells.

| SSM abrogated UVA-induced apoptosis in keratinocytes
UV irradiation can induce apoptosis in keratinocytes as a common manifestation of photoaging. 18To investigate the effect of SSM on UVA-irradiated HaCaT cells apoptosis, UVA-irradiated cells were pretreated with SSM.Annexin V-FITC/PI dual staining showed that UVA-induced HaCaT cell apoptosis was inhibited by SSM treatment (Figure 3A).Since the ratio of the pro-apoptotic protein Bax to the anti-apoptotic protein Bcl-2 can indicate apoptosis, 19 we detected Bax and Bcl-2 expression in different cell assays using western blotting.
The results showed that SSM pretreatment (20 and 40 μM) attenuated the UVA-induced overexpression of Bax.Compared with that of the untreated assay, the Bax/Bcl-2 ratio of the two SSM groups was even lower than that of the untreated assay (Figure 3B,C).These results indicate that SSM abrogated UVA-induced apoptosis in HaCaT cells.

| SSM reduced the expression of MMP-1 and MMP-9 in UVA-irradiated keratinocytes
The overexpression of MMPs, which are responsible for collagen degradation, is a characteristic of UVA-induced skin damage and indicating its potential for improving photodamage; however, the underlying mechanism requires further investigation.

| SSM inhibited UVA-induced oxidative stress in keratinocytes
Since ROS and oxidative stress play a major role in UV irradiationinduced cell damage, apoptosis, and even photoaging, the ROS

| SSM facilitated the antiphotodamage effects by downregulating ASK-1, JNK and p38 phosphorylation
The potential mechanism through which SSM protects HaCaT cells from UVA-induced photodamage was studied.The results showed that UVA irradiation increased the phosphate modification of ASK-1, JNK, and p38 in HaCaT cells compared to that in the control group, whereas SSM pretreatment significantly reduced the effects of UVA (Figure 6).In addition, 40 μM SMM exhibited stronger reversal efficacy than 20 μM SMM.These results suggest that the ASK/JNK/p38 pathway, which is involved in photoradical-induced cellular injury, is a regulatory target of SMM.

| DISCUSS ION
Sun exposure (or UV irradiation) is considered the most deleterious external factor impacting the skin, as it leads to reduced structural integrity, loss of physiological function, and ultimately photoinjury. 20,21Currently, phytochemicals are used to shield skin from photo-injury.With a broad range of pharmacological effects such as antioxidation, anti-inflammation, and immunomodulation, SSM has emerged as a potent lignan phytochemical. 13,22Nonetheless, only a limited number of studies have investigated the antiphotoaging effects of SSM on skin damage caused by UVA exposure.In the present study, SSM inhibited UVA-induced keratinocyte injury and ameliorated the expression of MMPs by reducing ROS formation.
The ASK-1-JNK/p38 MAPK pathway might be implicated in the regulatory process.Our results suggest that SSM is an ideal agent for protecting the skin from UVA irradiation.
At the cellular level, UVA causes photoaging by inducing ROS formation and subsequent oxidative damage to DNA, lipids, and proteins; if the damage is irreparable, death occurs via apoptosis. 23stologically, photoaged skin is characterized by a loss of collagen and induction of MMPs. 20,23In our study, the reduced cell viability and elevated expression levels of MMP-1 and MMP-9 indicated that UVA irradiation successfully causes cellular injury and photoaging in keratinocytes; however, SSM exhibited antiphotoaging effects.
4][25] Cells have developed a series of strategies to combat ROS, with superoxide and hydrogen peroxide being eliminated by antioxidant enzymes such as SOD and GSH peroxidase and/or nonenzymatic antioxidants such as vitamin E and GSH. 25   In our study, a high content of MDA, a lipid peroxidation marker overproduced by an increase in free radicals, 27 was observed in the UVA-irradiated model.We also observed reduced GSH content and SOD activity induced by UVA irradiation, confirming increased oxidative stress in the UVA-irradiated model.Sesamin pretreatment ameliorated these indicators of oxidative stress and UVA-induced intracellular ROS production.As a result of intracellular ROS accumulation, cells undergo apoptosis, as well as stimulated expression of MMPs. 20,28,29The Bcl-2 family, including Bcl-2 and Bax, regulate mitochondrial apoptosis when exposed to UV irradiation. 18Reactive oxygen species promote Bax-induced release of cytochrome c and activation of caspase-3, leading to intrinsic apoptosis pathways; Bcl-2 exerts opposite effects in this process. 30In our study, UVA irradiation caused a significant reduction in cell viability, and further investigation showed that apoptosis was involved in this effect.Sesamin pretreatment resulted in partial restoration of decreased apoptosis and increased MMP expression, demonstrating the antiphotodamage or antiphotoaging effects of SSM.These results suggest that SSM, as a photoprotective antioxidant, inhibits UVA-induced ROS formation and exerts protective effects in keratinocytes.
The MAPK signaling pathway, which is activated by ROS, participates widely in the regulation of cell apoptosis, proliferation, and differentiation. 31The relationship between SSM and MAPK signaling pathways has been extensively documented.These results provide an underlying molecular mechanism for our hypothesis that SSM counteracts ROS to alleviate keratinocyte injury, and consequently, photoaging.3][34] UVA activates MAPK pathways, particularly JNK, in human keratinocytes. 35JNK induces apoptosis through the activation of AP-1 that activates the expression of Fas/FasL signaling pathway-related proteins, or the alteration of mitochondrial membrane potential that results in the release of cytochrome c and the further activation of caspase 3. 36 p38 MAPK is significantly activated by UV irradiation, and the activated p38 will phosphorylate and contribute to the activation of numerous transcription factors, which orchestrate events leading to cell cycle arrest, DNA repair, and apoptosis. 37We observed that the intracellular ROS level and phosphorylation level of ASK-1, JNK, and p38 was increased by UVA irradiation and reversed by SSM pretreatment, indicating that UVA irradiation induced oxidative stress and activated the ASK-1-JNK/p38 signaling pathway in keratinocytes, while SSM counteracted the above signal transduction.ASK-1 activation occurs in numerous cellular responses, one of which is stress-and cytokine-induced apoptosis. 32,34,38ASK-1 is required for oxidative stress-induced sustained activations of JNK/p38 and consequently apoptosis. 39Moreover, adducts are formed between lipid peroxidation products and signal-regulating kinases such as p38 and JNK, which activate MAPKs, ultimately initiating the caspase-induced apoptotic process. 40The activation of both MAPK p38 and JNK leads to expression of AP-1, which in turn induces MMP-1, -3, and -9 expression, resulting in ECM degradation in keratinocytes responsible for photoaging. 3Taken together, this evidence supports our experiments on antioxidants and anti-apoptosis and ultimately the antiphotoaging effect of SSM and the underlying mechanism of the MAPK signaling pathway.We suggest that UVA induces keratinocyte injury and photoaging by activating the ROS-dependent ASK-1-JNK/ p38 death pathway, which was attenuated by SSM.Therefore, SSM shows potential resistance against photoaging (Figure 7).

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photoaging.20 Therefore, we first determined the expression of MMP-1 and MMP-9 using western blotting.The results suggested that 40 μM SSM pretreatment had a significant attenuative effect on the UVA irradiation-stimulated overexpression of MMP-1 and content and the levels of the oxidative stress biomarkers MDA, GSH, and SOD were measured in this study.UVA radiation significantly increased cellular MDA content and decreased GSH content and SOD activity, whereas all these indicators were significantly reversed by SSM pretreatment (Figure 5A-C).Subsequently, intracellular ROS production was determined using a DCFH-DA fluorescent probe.Sesamin pretreatment significantly reduced intracellular ROS levels in UVA-irradiated cells (Figure 5D), indicating that SSM pretreatment attenuates UVA irradiation-induced oxidative stress in HaCaT cells.F I G U R E 5 SSM inhibits UVA stimulated oxidative stress in keratinocytes.SSM reverses (A) the elevated MDA levels, (B) reduces GSH level, (C) decreases SOD expression, and (D) ROS formation induced by UVA in HaCaT cells.Statistical significance is set at ## p < 0.01 and ### p < 0.001 versus control group; **p < 0.01 and ***p < 0.001 versus the UVA-irradiated cell group.SSM, sesamin; UVA, ultraviolet A; HaCaT, human keratinocytes; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase.
Furthermore, intracellular thiols, GSH, and thioredoxin, which are responsible for maintaining strong reducing conditions in the cytoplasm, represent a major defense against ROS. 26 I G U R E 6 SSM mediates the antiphotodamage effects through inhibition of ASK-1, JNK, and p38 phosphorylation.(A) The content of p-ASK, p-JNK and p-p38 in UVA-irradiated HaCaT cells with or without SSM pretreatment is detected using western blotting.The relative protein expression of (B) p-ASK-1, (C) p-JNK, and (D) p-p38 is quantified using ImageJ.Statistical significance is set at ### p < 0.001 versus control group; ***p < 0.001 versus the UVA-irradiated cell group.HaCaT, human keratinocytes; SSM, sesamin; UVA, ultraviolet A; p-ASK-1, phospho-apoptosis signal-regulating kinase-1; p-JNK, phospho-c-Jun N-terminal protein kinase.
photoprotective botanical agent in the treatment of photoaging and as a new candidate for cosmetic formulations against photodamage.