Curcumin Improves Keratinocyte Proliferation, Inflammation, and Oxidative Stress through Mediating the SPAG5/FOXM1 Axis in an In Vitro Model of Actinic Dermatitis by Ultraviolet

Background Chronic actinic dermatitis (CAD) is an abnormally proliferating photoallergic skin disease. Dysregulated inflammation and oxidative stress are the immediate factors in the abnormal proliferation of keratinocytes. This study aimed to investigate the effect of curcumin on the aberrant proliferation of keratinocytes in an in vitro (actinic dermatitis) AD model and the possible molecular mechanisms. Methods The keratinocytes were irradiated with ultraviolet (UV) to construct an in vitro AD model and then processed with different concentrations of curcumin. Cell viability, oxidative stress markers (SOD, GSH-PX, and MDA), activated oxygen species (ROS), and inflammation markers (IL-1β, IL-6, IL-18, and TNFα) were determined, respectively. Western blot was applied to assay the profiles of apoptosis-related proteins (Bax, Bcl-xL, Caspase3, Caspase8, and Caspase9), oxidative stress proteins (Keap1, Nrf2, HO-1, COX2, and iNOS), and inflammatory proteins (NF-κB, MMP1, and MMP9) and SPAG5/FOXM1. Functionally, SPAG5 or FOXM1 overexpression and knockdown models were constructed in keratinocytes to characterize their influence on UV irradiation-mediated keratinocyte dysfunction. Results Curcumin weakened UV-mediated inflammation, proliferation, and oxidative stress and impaired apoptosis in keratinocytes. UV boosted SPAG5/FOXM1 expression in cells, while curcumin concentration-dependently retarded SPAG5/FOXM1 expression. Overexpression of SPAG5/FOXM1 fostered UV-mediated inflammation, proliferation, oxidative stress, and intensified apoptosis, whereas curcumin mostly reversed the SPAG5/FOXM1-mediated effects. In addition, knocking down SPAG5/FOXM1 ameliorated UV-mediated keratinocyte dysfunction, whereas curcumin failed to exert further protective effects in cells with knockdown of SPAG5/FOXM1. Conclusion Curcumin modulated proliferation, inflammation, oxidative stress, and apoptosis of keratinocytes by restraining the SPAG5/FOXM1 axis.


Introduction
Chronic actinic dermatitis (CAD) is a photoallergic disorder caused by ultraviolet A (UVA), ultraviolet B (UVB), or visible light [1]. CAD is most often seen in middle-aged and older men who have been exposed to the sun for many years and is typically characterized by pruritic eczema and mossy plaques with markedly reduced skin folds in sun-exposed areas, including the chin, the area behind the ears, and the nasolabial folds [2]. The treatment of CAD requires avoidance of light and exposure to well-defined allergenic substances and medications, strict sun protection when going out, and sometimes the necessity to avoid outdoor activities altogether, which seriously affects the patients' quality of life [3,4]. There is no curative treatment for CAD currently [5]; it is imperative to develop novel strategies for CAD treatment.
Curcumin is the predominant active ingredient in Curcuma longa with pronounced anti-inflammatory, antioxidant, antitumor, anticoagulant, and anti-infective effects [6]. As reported, curcumin mitigates oxidative stress and apoptosis in UVB-irradiated HaCaT cells by diminishing reactive oxygen species (ROS) levels and curbing the mitochondrial pathway [7]. Curcumin exhibits antiinflammatory effects in acute skin inflammation by restraining COX-2 expression in UVB-irradiated human keratinocytes (HaCaT) through blocking the activity of p38 MAPK and JNK [8]. In parallel, curcumin restricts UVB-induced growth and proliferation of keratinocytes by enhancing p53 phosphorylation through the activation of p38 MAP kinase [9]. Nonetheless, the molecular mechanisms by which curcumin alleviates ultraviolet-(UV-) mediated keratinocyte dysfunction await further investigation.
Sperm-associated antigen 5 (SPAG5) is a mitotic spindle protein that is implicated in cell proliferation, apoptosis, and autophagy [10]. Increased SPAG5 expression is linked to unfavorable prognosis in glioma patients. Knockout of SPAG5 restrains proliferation, colony formation, migration, and invasion of glioma cells and stimulates apoptosis [11]. Forkhead box M1 (FOXM1) belongs to the Forkhead family, which is a transcription factor associated with cell proliferation [12]. Low-power laser irradiation (LPLI) delays UVBinduced cellular senescence by activating extracellular regulated protein kinases (ERKs) to facilitate the phosphorylation of FOXM1 and nuclear translocation to downregulate p21 [13]. Previous studies have revealed that SPAG5 silencing decreases matrix metalloproteinase-2 (MMP2) expression by downregulating FOXM1 to curb osteosarcoma (OS) cell invasion, migration, metastasis, and epithelialmesenchymal transition (EMT) [14]. Nevertheless, the mechanism of action of SPAG5/FOXM1 in CAD is elusive.
In the present study, HaCaT cells were subject to UV irradiation to develop an in vitro actinic dermatitis (AD) model and discovered that curcumin abated the proliferation, inflammation, oxidative stress, and apoptosis of UV-irradiated keratinocytes and hindered the SPAG5/FOXM1 pathway. Overexpression of SPAG5/FOXM1 worsened UV irradiation-mediated keratinocyte dysfunction, which was ameliorated by knockdown of SPAG5/FOXM1. In conclusion, curcumin exerts keratinocyte-protective effects via repression of the SPAG5/FOXM1 pathway in an in vitro AD model.

UV Irradiation and Drug Treatment.
The medium was substituted with phosphate-buffered saline (PBS, 200 μL/ well), and HaCaT cells were then exposed to UVB radiation (range: 280-315 nm; dose: 50 mJ/cm 2 ; duration: 45 min) with the aid of a BIO-LINK crosslinker (BLX; Vilber Lourmat, Collégien, France). Following UVB irradiation, the cells were flushed with PBS and cultured for 24 hours in a fresh medium. UVB-induced HaCaT cells were then processed with curcumin (2.5, 5 μM) for 24 hours.
2.4. The Cell Counting Kit-8 (CCK-8) Assay. The CCK-8 experiment was used to determine cell viability. HaCaT cells from each treatment group were inoculated in 96-well plates at 1 × 10 3 cells per well. After 24 hours of incubation, 10 μL Table 1: Primers used in this study.

Disease Markers
were cleared with serum-free DMEM and the fluorescence intensity was checked with a fluorescent microscope (Leica, Germany). Finally, the ROS level was determined with the fluorescence level.

The Detection of SOD, GSH-PX, and MDA Levels.
HaCaT cells were inoculated in 6-well plates at 1:5 × 10 5 /per well and incubated overnight. Then, the cells were collected from each treatment group, flushed, and lysed in liquid nitrogen. The levels of SOD, GSH-PX, and MDA in the cells were estimated using the Oxidative Stress Assay Kit (Beyotime, Shanghai, China) based on the manufacturer's description.

Statistics and Analysis.
Experimental results were presented as mean ± SD. Data analysis was performed using the SPSS 22.0 software (IBM, Armonk, NY, USA). The measures were analyzed by ANOVA, and statistical significance was determined by t-test or ANOVA. The two-sided p < 0:05 was regarded as statistically significant.  Figure 1(e)). As testified by RT-PCR outcomes, UV irradiation enhanced the expression of inflammatory factors IL-1β, IL-6, IL-18, and TNFα in HaCaT cells versus the Con group, and curcumin processing dampened the expression of these inflammatory factors in UV-irradiated HaCaT cells (Figure 1(f)). WB results displayed elevated expressions of Bax, Caspase3, Caspase8, Caspase9, Keap1, COX2, iNOS, NF-κB, MMP1, and MMP9 and diminished expressions of Bcl-xL, Nrf2, and HO-1 in UV-irradiated HaCaT cells compared to the CON group, and curcumin treatment reversed these effects (Figures 1(g)-1(i)). In addition, we observed that the effect of 5 μM curcumin was obviously stronger than       (Figure 4(a)). Next, we treated transfected HaCaT cells with UV irradiation and then with 5 μM curcumin for 24 hours. As disclosed by CCK-8 outcomes, overexpressing FOXM1 enhanced the proliferative viability of UVirradiated HaCaT cells versus the UV group. The proliferation of HaCaT cells was restricted in the FOXM1 + UV + curcumin group compared to the FOXM1 + UV group (Figure 4(b)). The results of the oxidative stress assay kit and cytofluorimetry hinted that the levels of SOD and GSH-PX were reduced and the contents of MDA and ROS were uplifted in UV-irradiated HaCaT cells after overexpression of FOXM1. In parallel, as compared to the FOXM1 +      15 Disease Markers cells without affecting the expression of SPAG5 significantly, whereas curcumin treatment abated these effects (Figures 4(h)-4(k)). These results hinted that overexpressing FOXM1 contributed to proliferation, inflammation, oxidative stress, and apoptosis in UV-irradiated keratinocytes, whereas curcumin largely reversed the effects mediated by FOXM1 overexpression.

Knocking Down SPAG5 Ameliorated UV-Irradiated
Keratinocyte Dysfunction. To further exploit the role of SPAG5 in AD, we transfected si-NC and si-SPAG5 into HaCaT cells. The SPAG5 expression in transfected HaCaT cells was checked by RT-PCR after 24 hours, which displayed that SPAG5 was remarkably downregulated in HaCaT cells in the si-SPAG5 group versus the si-NC group ( Figure 5(a)). We then exposed the transfected HaCaT cells to UV irradiation and treated them with 5 μM curcumin for 24 hours. The CCK-8 assay illustrated that knocking down SPAG5 curbed the proliferative viability of UVirradiated HaCaT cells. Treatment with curcumin based on the si − SPAG5 + UV group had no remarkable impact on HaCaT cell proliferation ( Figure 5(b)). The results of the oxidative stress assay kit and cytofluorimetry disclosed that knockdown of SPAG5 heightened the levels of SOD and GSH-PX and hindered the contents of MDA and ROS in UV-irradiated HaCaT cells compared to the UV group. In parallel, the levels of SOD, GSH-PX, MDA, and ROS were not significantly altered in HaCaT cells in the si − SPAG5 + UV + curcumin group versus the si − SPAG5 + UV + curcumin group (Figures 5(c)-5(f)). As demonstrated by RT-PCR findings, knockdown of SPAG5 restrained the expression of inflammatory factors IL-1β, IL-6, IL-18, and TNFα in UV-irradiated HaCaT cells. Meanwhile, curcumin treatment did not influence the expression of inflammatory factors in UV-irradiated HaCaT cells after knockdown of SPAG5 compared to the si − SPAG5 + UV group ( Figure 5(g)). Also, WB results confirmed that knockdown of SPAG5 downregulated Bax, Caspase3, Caspase8, Cas-pase9, Keap1, COX2, iNOS, NF-κB, MMP1, MMP9, SPAG5, and FOXM1 and upregulated Bcl-xL, Nrf2, and HO-1 in UV-irradiated HaCaT cells compared to the UV group. Nevertheless, there was no significant difference in the expression of the above proteins in HaCaT cells in the si − SPAG5 + UV group versus the si − SPAG5 + UV + curcumin group (Figures 5(h)-5(k)). Thus, knockdown of SPAG5 alleviated UV irradiation-mediated keratinocyte dysfunction.

Knockdown of FOXM1 Eased UV Irradiation-Mediated
Dysfunction of Keratinocytes. To further investigate the function of FOXM1 in AD, we transfected si-NC and si-FOXM1 into HaCaT cells. The transfection efficiency was checked by RT-PCR after 24 hours, which highlighted that the expression of FOXM1 was declined in HaCaT cells

20
Disease Markers after transfection with si-FOXM1 compared to the si-NC group (Figure 6(a)). Next, we treated the transfected HaCaT cells with UV irradiation and then with curcumin (5 μM) for 24 hours. As uncovered by CCK-8 results, the proliferation of HaCaT cells was impaired in the si − FOXM1 + UV group versus the UV group. Meanwhile, curcumin treatment did not influence the proliferation of UV-irradiated HaCaT cells after knockdown of FOXM1 versus the si − FOXM1 + UV group (Figure 6(b)). Oxidative stress assay kits and cytofluorimetric assays demonstrated that knockdown of FOXM1 elevated SOD and GSH-PX levels and

Discussion
CAD is an immune-mediated photodermatosis. As the disease progresses, the lesioned skin becomes rough and thickened, with mossy lesions and increased pruritus, and patients may even scratch the skin and suffer complications such as local skin infections and artificial dermatitis [15,16].
Here, we demonstrated that curcumin diminishes proliferation, inflammation, oxidative stress, and apoptosis of keratinocytes via repression of the SPAG5/FOXM1 axis in an in vitro AD model. UV is the light in sunlight with a wavelength of 10 nm to 400 nm and can be classified as UVA, UVB, and UVC. UV is not visible as it lies outside of the violet light in the spectrum [17]. Excessive exposure of the skin to UV radiation causes oxidative stress, inflammation, immunosuppression, matrix metalloproteinase production, and DNA damage and mutations, resulting in skin photodamage [18]. Corneocytes, the main functional and structural component of the epidermis, account for more than 80% of epidermal cells and are the first line of defense against UV radiation [19,20]. Here, we constructed an in vitro AD model in UV-irradiated HaCaT cells and observed that UV irradiation dramatically stimu-lated oxidative stress and inflammatory responses in HaCaT cells compared to controls.
Curcumin is a natural compound isolated from Curcuma longa, which exerts an essential role in inflammatory, neoplastic, and infectious skin diseases [21]. For instance, curcumin protects HaCaT cells from UV irradiation-induced photodamage by activating nuclear factor erythroid 2-related factor 2 (Nrf2) to restrain apoptosis and oxidative stress [22]. Curcumin protects the skin from UVB-induced epidermal cytotoxicity by triggering the Keap1-Nrf2 pathway [23]. Curcumin suppresses oxidative stress in acute UVB-irradiated immortalized human keratinocytes (HaCaT) through activation of Nrf2 and exerts a photoprotective effect in acute photodamage [24]. Our findings are concordant with previous studies where we discovered that curcumin treatment substantially increased the proliferation, inflammation, oxidative stress, and apoptosis of UVirradiated keratinocytes with higher concentrations of curcumin having a more pronounced effect.
SPAG5 is an emerging oncogene that is overexpressed and exerts carcinogenic effects in diverse cancers such as lung adenocarcinoma [25] and liver cancer [26]. In diabetic nephropathy, lncRNA SPAG5-AS1 activates the AKT/ mTOR pathway through upregulation of SPAG5 to foster apoptosis and abate autophagy in high glucose-(HG-) treated human podocytes (HPCs) [27]. FOXM1 is a novel tumor biomarker that is involved in modulating psoriasis [28], keloid [29], and many other skin diseases. FOXM1 is notably upregulated in psoriatic skin tissues and in TNFαstimulated HaCaT cells. FOXM1 knockout curbs TNFαinduced proliferation and inflammatory response of HaCaT cells and potentiates apoptosis by restraining the NF-κB pathway [30]. A study has revealed that downregulation of SPAG5 suppresses the ADAM17/NOTCH1 pathway by lowering the expression of FOXM1 in malignant melanoma (MM), thereby dampening the viability, migration, invasion, and EMT of MM cells [31]. Nevertheless, the involvement of the SPAG5/FOXM1 pathway in AD is unclear. In the present research, we identified that UV contributed to the expression of the SPAG5/FOXM1 pathway, while curcumin concentration-dependently restrained the SPAG5/FOXM1 pathway. Overexpression of SPAG5/FOXM1 facilitated UV irradiation-mediated dysfunction in keratinocytes, whereas curcumin mostly reversed the effects mediated by SPAG5/ FOXM1 overexpression. Knockdown of SPAG5/FOXM1 ameliorated UV irradiation-mediated keratinocyte dysfunction and counteracted the protective effect of curcumin on keratinocytes.
Overall, our study illustrates that curcumin protects keratinocytes from UV irradiation-induced photodamage by modulating the SPAG5/FOXM1 axis in an in vitro AD model. Our findings shed light on new ideas and directions for the treatment of AD.

Data Availability
The data will be available on reasonable requests from the corresponding author.