4‐phenylpyridine suppresses UVB‐induced skin inflammation by targeting c‐Src in vitro and in vivo

Abstract Acute or repetitive exposure to ultraviolet (UV) cause disruptions to the skin barrier and subsequent inflammatory skin disease. 4‐phenylpyridine (4‐PP) is a constituent of Brassica campestris L. ssp. Pekinensis and its effect on skin inflammation and molecular target remain unclear. The purpose of this study is to confirm the anti‐inflammatory efficacy of 4‐PP on UVB‐induced skin inflammation in human keratinocytes HaCaT and mouse skin and validation of its molecular target. 4‐PP also attenuated UVB‐induced phosphorylation of p38/mitogen‐activated protein kinase kinase (MKK) 3/6, c‐Jun N‐terminal kinase 1/2, MKK 4/7, extracellular‐signal‐regulated kinase 1/2, mitogen‐activated protein kinase 1/2. Additionally, 4‐PP inhibited UVB‐induced phosphorylation of epidermal growth factor receptor (EGFR) Y1068, Y1045 and 854 residues but not the proto‐oncogene tyrosine‐protein kinase c‐Src. Drug affinity responsive target stability assay revealed that 4‐PP directly binds to c‐Src and inhibits pronase c‐proteolysis. Knockdown of c‐Src inhibited UVB‐induced COX‐2 expression and phosphorylation of MAPKs and EGFR in HaCaT cells. Dorsal treatment of 4‐PP prevented UVB (0.5 J/cm2)‐induced skin thickness, phosphorylation of EGFR and COX‐2 expression in mouse skin. Our findings suggest that 4‐PP can be used as anti‐inflammatory agent with an effect of skin inflammation by inhibiting the COX‐2 expression via suppressing the c‐Src/EGFR/MAPKs signalling pathway.

UVB is considered as carcinogenic as exposure to these wavelengths alone resulted in skin cancer in SKH-1 hairless mice. 5 COX-2 is an inducible enzyme that catalyses the rate-limiting conversion of prostaglandin E2 (PGE 2 ), a critical mediator in inflammation. 6 UVB-induced COX-2 has a vital role in skin inflammation and cancer. Celecoxib, a COX-2 inhibitor, suppressed UVB-induced skin inflammation such as skin thickness, PGE 2 production, sunburn and carcinogenesis in vivo. 7 Inhibition of COX-2 expression increased by UV irradiation also suppressed the development of skin inflammation, photoageing and skin cancer. 8 Signalling molecules mitogen-activated protein kinases (MAPKs) and activated protein (AP)-1 transcriptional factor directly regulate UVB-induced COX-2 expression in HaCaT and SKH-1 hairless mice. 5 Although the cell receptor for UV has not been fully elucidated, epidermal growth factor receptor (EGFR) links UV-induced MAPKs phosphorylation and AP-1 activity. A previous study reported that after UVB radiation, c-Src encourages the internalization of active EGFR, which in turn leads to the activation of the ERK1/2 pathway, resulting in increased COX-2 expression. 9 The c-Src inhibitor, PP2, inhibited UVB-induced COX-2 expression and phosphorylation of ERK, p38, c-Jun N-terminal kinase (JNK), Akt and EGFR (Y845) compared with scrambled control in JB6P+ cells. 10 Therefore, inhibition of c-Src and EGFR signalling pathway could be a therapeutic target to alleviating UV-induced skin inflammation.
Botanical extracts are frequently used as traditional remedies for inflammation. Anti-inflammatory effects of extracts and phytochemicals in fruits, herbs and vegetables have been reported in skin-related cells and mice. 11 4-phenylpyridine (4-PP) is a volatile compound present in Brassica campestris L. ssp. pekinensis known as Chinese cabbage and/or Korean cabbage. 12 Although it is reported that Chinese cabbage exerts antioxidant, antimicrobial and antiatherosclerosis properties. 13

| Cell culture, UVB exposure and cell viability
Human epidermal keratinocyte HaCaT cells were cultured in DMEM containing 10% FBS, and 1% penicillin-streptomycin at 37°C and 5% CO 2 in a humidified incubator (Thermo Fisher Scientific,).
HaCaT cells were grown to 70%-80% confluency in a 96-well plate and pretreated with 4-PP dissolved in dimethyl sulfoxide For in vitro study, we replaced with serum free before sample treatment, cultured for 24 h, and then conducted the further experiments.

| Animal experiments
5-weeks-old male ICR mice were purchased from Joongah Bio (Joongah Bio,). The animals were housed in climate-controlled quarters (25°C at 50% humidity) with a 12-h light/12-h dark cycle. All animals received humane care, and the study protocol (2020-0007) was approved and performed in accordance with guidelines for animal use and care at Kyungpook National University. Animals were stabilized for 1 week prior to the study with free access to food and water. For animal study, acetone was used as vehicle and 4-PP was dissolved in acetone. Twenty mice were randomly allocated to each group (n = 5 per each group, four groups in total): (1) control group

| AP-1 promoter assay
HaCaT cells (1 × 10 5 cells/ml) stably infected with pGreeFire plasmid containing MMP-1 promoter were grown to 70%-80% confluency in 96-well plates. The media was replaced with serum-free DMEM for   The band intensity of western blot was analysed using image J software program (National Institutes of Health,).

| Cytosolic and nuclear fractions
HaCaT cells (1 × 10 5 cells/ml) were grown to 70%-80% confluency in 10 cm dishes. The media was replaced with serum-free DMEM for 24 h and 4-PP added at 12.5, 25 and 50 μM for 1 h. After UVB irradiation (0.03 J/cm 2 ), cells were incubated for 30 min, collected with PBS and separated with the NE-PER™ Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific,). Cell extracts separated into nucleus and cytoplasm were assessed via western blot assay.

| Small interfering RNA knockdown assay
HaCaT cells were grown to 50%-60% confluence in 10 cm dishes.  and 50 μM. Cell viability was measured by the MTS assay. The data represent the mean ± SD of three independent experiments. #p < 0.05 between the control group and the group exposed to UVB alone; **p < 0.01 and ***p < 0.001 between groups irradiated with UVB and 4-PP and the group exposed to UVB alone

| Histological analysis
Mouse skin separated from back was embedded with OCT solution in frozen section. Frozen section was cut into 6μm-thick sec- The thickness of the epidermis and the COX-2 were visualized using a fluorescent microscope (Leica microsystems,) and images were analysed using Leica Application Suite X (Leica microsystems) software.

| Statistical analysis
Where appropriate, data are expressed as the mean ± standard deviation (SD), and significant differences between UVB and 4-PP groups were calculated with one-way anova with LSD post-hoc test (analysis of variance) of IBM SPSS software (IBM Corp.,). Student's t-test was used to assess significance of differences between the groups. A probability value of p < 0.05 was used as the criterion for statistical significance.

COX-2 is a critical factor in UV-caused skin inflammation and could
be an effective target for regulation of skin diseases. 15 Therefore, we first investigated the effect of 4-PP on UVB-induced COX-2 expression in HaCaT cells. 4-PP significantly inhibited UVB-induced COX-2 expression and cox-2 mRNA in HaCaT cells ( Figure 1B,C). PGE 2 , a product of COX-2, induces acute inflammation and inflammatory disorders. 16 4-PP significantly inhibited UVB-induced PGE 2 production in HaCaT cells ( Figure 1D). Additionally, we observed that 4-PP significantly inhibited UVB-induced AP-1 activity in HaCaT cells

F I G U R E 2 The effect of 4-PP on UVB-induced phosphorylation and nuclear translocation of c-Jun in HaCaT cells. (A) 4-PP inhibits UVB-induced phosphorylation of c-Jun in HaCaT cells. (B) and (C) 4-PP inhibits UVB-induced translocation of c-Jun in HaCaT cells; N, nuclear fractions; C, cytosol fractions.
Cells were pretreated with 4-PP for 1 h, treated with UVB and harvested after 30 min. Phosphorylation of c-Jun in whole lysate and c-Jun expression in the nuclear and cytoplasmic fractions was analysed by western blot assay. Cytosolic and nuclear fractions and immunofluorescence were described in the Materials and Methods section. The data represent the mean ± SD of three independent experiments. #p < 0.05 between the control group and the group exposed to UVB alone; **p < 0.01 and ***p < 0.001 between groups irradiated with UVB and 4-PP and the group exposed to UVB alone ( Figure 1E). 4-PP was non-toxic at 12.5, 25 and 50 μM in HaCaT cells ( Figure 1F).

| 4-PP inhibits UVB-induced phosphorylation of MAPKKs/MAPKs in HaCaT cells
MAPKs are representative signalling molecules in the regulation of UV-medicated AP-1 transcriptional activity. 18 Figure 3B). However, 4-PP did not affect the phosphorylation of Akt ( Figure 3C).  Figure 4A). c-Src is known to regulate EGFR activity by phosphorylation of EGFR at Tyr 845. 19 Therefore, we measured the effect of 4-PP on UVB-induced phosphorylation of EGFR at Tyr 845 and demonstrated that 4-PP inhibited phosphorylation at this residue ( Figure 4B). Since 4-PP inhibits UVB-induced activation of c-Src-specific downstream regulator, we hypothesized that 4-PP may affect EGFR activity via direct binding to c-Src. To confirm our hypothesis, we conducted a DARTS assay and revealed that 4-PP increased c-Src stability against pronase treatment ( Figure 4C).

| Knockdown of c-Src inhibits UVBinduced COX-2 expression and phosphorylation of MAPKs and EGFR in HaCaT cells
To confirm the role of c-Src on UVB-induced COX-2 expression and MAPKs signalling pathways, we transfected HaCaT cells with siRNA for c-Src knockdown. Knockdown of c-Src mRNA inhibited UVB-induced phosphorylation of EGFR at Tyr845 ( Figure 5A). Additionally, Knockdown of c-Src inhibited UVB-induced COX-2 expression ( Figure 5B) and phosphorylation of MAPKs including p38, ERK1/2, JNK1/2 ( Figure 5C).

| 4-PP inhibits UVB-induced epidermal thickness, COX-2 expression and phosphorylation of EGFR (Y845) in ICR mice
We then measured the inhibitory effect of 4-PP on UVB-induced skin inflammation in vivo. 4-PP treated mice showed a significant reduction in epidermal thickening compared to the UVB irradiationonly mice ( Figure 6A). Western blot assay with protein extract from mouse skin showed that UVB irradiation of the dorsal skin of mice up-regulated phosphorylation of EGFR (Y845) and COX-2 expression F I G U R E 4 The effect of 4-PP on the UVB-induced phosphorylation of c-Src and EGFR in HaCaT cells. (A) 4-PP inhibits the UVB-induced phosphorylation, EGFR (Y1068) and EGFR (Y1045) in HaCaT cells, but had no effect on phosphorylation of c-Src. (B) 4-PP inhibits the UVBinduced phosphorylation of EGFR (Y845) in HaCaT cells. Cells were pretreated with 4-PP for 1 h, irradiated with UVB, and then harvested after 15 min. Phosphorylation and expression levels were detected by western blot assay with specific antibodies. The data represent the mean ± SD of three independent experiments. #p < 0.05 between the control group and the group exposed to UVB alone; **p < 0.01 and ***p < 0.001 between groups irradiated with UVB and 4-PP and the group exposed to UVB alone. (C) 4-PP directly binds to c-Src in HaCaT cells. For the DARTS assay, cells lysate was treated with 4-PP at the indicated concentrations for 1 h and were digested with pronase and assessed via western blot assay. The data represent the mean ± SD of three independent experiments. **p < 0.01 between groups treated with pronase and 4-PP and the group treated with pronase only and that this abnormal expression was significantly suppressed by 4-PP ( Figure 6B,C). Additionally, immunofluorescence results also showed that treatment of 4-PP suppressed UVB-induced COX-2 expression in mouse skin ( Figure 6D).

| DISCUSS ION
The epidermis of skin enables essential photoprotection, adaptive immunity and absorption of harmful chemicals. 20  The data represent the mean ± SD of three independent experiments. #p < 0.05 between the control group and the group exposed to UVB alone;**p < 0.01 and ***p < 0.001 between groups treated with scrambled siRNA and UVB and groups treated with c-Src siRNA and UVB harmful environmental factor and causes skin inflammation and development of skin cancer. 21 Skin damage caused by UV exposure is important as a pre-stage of disease development, and can also cause anxiety due to effects on physical appearance.
Recently, cosmeceuticals, cosmetic with added pharmacological efficacy, have received increased attention. 22 Previously, we proved that natural compounds could be promising cosmeceutical materials with preventive capacity for UVB-induced skin inflammation and skin cancer. 5,8 Cruciferous vegetables are promising anti-inflammatory agents, and we previously screened Phosphorylation and expression levels of mice dorsal skin were detected by western blot assay. The data represent the mean ± SD of three independent experiments. # p < 0.05 between the control group and the group exposed to UVB alone; *p < 0.05, **p < 0.01 and ***p < 0.001 between groups irradiated with UVB and 4-PP and the group exposed to UVB alone related to skin inflammation and finally development of skin cancer. 25 Therefore, we examined the effect of different concentrations several studies reported that active c-Src directly phosphorylates EGFR at tyrosine 845. 29 In our western blot assay, we found that 4-PP inhibited UVB-induced phosphorylation of EGFR at tyrosine 845; however, 4-PP did not affect UVB-induced phosphorylation of c-Src, and therefore we hypothesized that 4-PP directly affects phosphorylation of EGFR at tyrosine 845 by direct binding to c-Src.
The DARTS assay can be used to prove the interaction between compound and target protein by using a proteinase, such as pronase. 30 Using the DARTS assay, we evaluated the interaction be-

| CON CLUS ION
In this study, we confirmed that 4-PP, a compound found in Chinese

CO N FLI C T O F I NTE R E S T
There is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.